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The University of New Mexico

THE UNIVERSITY OF NEW MEXICO

COLLEGE OF PHARMACY

ALBUQUERQUE, NEW MEXICO

Correspondence Continuing Education Coursesfor

Nuclear Pharmacists and Nuclear Medicine Professionals

VOLUME II, NUMBER 2

Radiopharmaceuticals for Imagingof

Infectious and Inflammatory Lesions

by:

Joseph C. Hung, R.Ph., Ph.D.

Manuel L. Brown, M.D.

Co-sponsored by: mpi~rmcg services incan -ersham company

m Tht University of New Mexico Collsge of Pharmacy is approved by the American Council on Pharmaceutical

Educa[ion as a provider of continuing pharmaceutical education. Program No. 180-039-93-006. 2,5 Contact

Hours or .25 CEU”S

@

.

and

Director of Ptiwcy Coti”nuing Education

William B. Hladik III, M. S., R.Ph.College of Pharmacy

University of New Mexico

AssoczizteEditor

Production Specialist

Sharon I. Rarnirez, Staff AssistantCollege of Pharmacy

University of New Mexico

While the advice and information in this publication are believed to be true and accurate at press time, neither the author(s) northe editor nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publishermakes no warranty, express or implied, with respect to the material contained herein.

Copyright 1993University of New Mexico

Pharmacy Continuing EducationAlbuquerque, New Mexico

RADIOPHARMACEUTICALS FOR IMAGING OFINFECTIOUS AND INFLAMMATORY LESIONS

OF OBJECTIVES

The primary goal of [his corrcspondcncc course is to incrcasc the reader’s knowldgc and understanding of the variousradiopharmaccuticals USC(,Ifor localizing in fwtious and inflammatory lesions. In order to achieve this goal, this continuing education lessonpresents pertinent information for undcrsbnding mclhods of production, the mechanisms for labeling and localization, and the physical,

chemical, and biological properties of different imaging rtidiopharmaccuticals for infection and/or inflammation imaging. In addition,advantages and disadvantages of wch infection/inflammation imaging radiopharmticcutical as well as comparisons among differentradiopharmaccuticals arc also discussed. The conclusion will include a discussion of clinical application of the radiopharmaccuticals usdin detecting and evaluating infectious and inflammatory lesions.

Upon successful completion of thiv &erial, the reader shouti be abfe to:

1.

3.

4.

5.

6.

0 7+

8.

9.

10.

11.

12.

13.

14.

15,

16.

17.

●18.

19+

20.

Identify the diffcrcnccs bctwccn infection and inflammation.

Discuss the advantages and disadvantages of different modalities used for localizing infectious and inflammatory lesions.

List the required characteristics of an ideal imaging radiopharmaccutical for detecting infectious and inflammato~ lesions.

Describe the currently accepted mechanisms of localization for gallium-67 (b7Ga) citrate,

Discuss the distinctive characteristics and functions of five major types of white blood cells (WBCS),

Describe the general steps involved in the method for radiolabeling leukocytes.

Compare the differences between using pure granulocytcs and mixed leukocytes in infection/inflammation imaging studies.

State the various factors that may aff-t the labeling efficiency of radiolabeled WBCS.

Describe the in vitro and in vivo quality control procedures for radio labeled leukocytes.

List several drawbacks associated with technetium-99m (WmTc)-labeled leukocytes using both the phagocytic and pretinning

methods.

List the advantages and disadvantages of using iridium-111 (11‘In)-labeled leukocytes as an imaging agent for the detection ofinfectious and/or inflammatory disease.

Present the proposed mechanism involved in the labeling process for 11IIn-oxine-labeld leukocytes.

Explain the circumstances which necessitate a search for other new completing agents of iridium other than oxine.

Compare and contrast the biologic properties between *Tc hexamethylpropylene amineoxime (HMPAO)-labeled leukocytesand 11IIn.oxinc labeled leukocytes.

Describe the clinical advantages and potential limitations associated with the use of radiolabeled leukocytes for imaginginfection/inflammation sites.

List five major difficulties related to the use of radio labeled antibodies for the detection of infection and/or inflammation.

Describe the advantages and disadvantages involved with the clinical use of “’In-labeled nonspecific polyclonal humanimmunoglobulin (lgG) for the detection of infectious and/or inflammatory foci.

List the underlying principles for ““Tc-labeled BW 250/183 when used as an infection/inflammation imaging agent.

Compare the size of ~nTc nanocol]oids with other particulate imaging radiopharmaceuticals for infection and/or inflammation

detection.

Discuss the approach for improvement of the target-to-nontarget rtitios in the clinical use of lllIn-label~ avidin-biotin.

1

COURSE OUTLINE RADIOPHARMACEUTICALS FOR IMAGING OFlNFECTIOUS AND INFLAMMATORY LESIONS

1. INTRODUCTIONA. Pathopllysiology of Infection find Inflammation

B. Modalities for Localti/.~tion of Infection andInflammation

c. Ideal Characteristics of Radiopharmaccuticalsfor Imaging Infectious and InflammatoryLesions

D. Radiophartnaccuticals for the Detection ofInfectious and Inflammatory Lesions

11. RADIONUCLIDESA. ‘7Ga-CitrateB. l“lnCil

II. RAD1OLABELED LEUKOC~ESA. Biological Functions of LeukocytesB, General Steps for Radiolabeling Leukocytes

1, Collection of Blood?-, Separation of “Crude” Leukocytes

3. Separation of “Pure” Granulocytesa. Pure Granulocytes vs.

Crude Leukocytes

4. Cell Labelingc. Factors Which Affect Labeling EfficiencyD. Quality Control of Radiolabeled Leukocytes

1. In Virro Tests9-. In Vivo Tests

3. Leukocyte Count

E. IIlIn-Labe]d Leukocytes

1. Mwhanism of Leukocyte LabelingWith 1llln-Oxine

9-. Biological Distribution of 1IlIn.Oxine.Labeled Leukocytes

3. Dosage

4. Imaging Protocol

5. 1“In and Other Chelating Complexes

F. *T~-Labeld Leukocytes

1. Leukocytes Labeled with *Tc UsingPhagocytic and Pretinning Methods

-) %Tc.HMpAO-Labeld LeukocYte~-.a. Biodistribution of ‘Tc.

HMPAO -LabeledLeukocytes

b: Elution of *Tc-HMPAOActivity

c. Selective Radiolabeling

G. Potential Pitfalls Associated With RadiolabeledLeukocytes

Iv. RADIOLABELED ANTIBODIESA. 1~~In.Label~d Nonspwiflc polyclonal Human

Immunoglobulin

B. ‘Tc-Labeled Monoclinal Antigranulocyte

Antibody (%Tc-Labeled BW 250/183)

v. OTHER AGENTS

A< ‘Tc-Labeled NanocolloidsB. IIlIn.Labeled Avidin-Biotin

VI. CONCLUSIONS

By:

Joseph C. Hung, R.Ph., Ph.D.Assistant Professor of Radiology

Mayo Medical SchoolDirector of Nuclear Pharmacy

Nuclear MedicineDepartment of Diagnostic Radiology

Mayo Clinic200 First Street S.W.Rochester, MN 55905

Manuel L. Brown, M.D.Professor, Diagnostic Radiology

University of Pittsburgh School of MedicineChief, Division of Nuclear Medicine

Department of RadiologyUniversity of Pittsburgh Medical Center

DeSoto at O’Hara StreetsPittsburgh, PA 15213

INTRODUCTION

Pathophysiology of Infection and InflammationInfection involves the invasion and multiplication of

microorganisms in body tissues which may result in localtissue injury or destruction, In order to destroy, dilute,or sequester the invading microorganisms and injuredtissue, the body’s defense mechanism responds with acomplex series of events. It is normally associated inthe acute stage with signs of inflammation: heat (calor),redness (rubor), swelling (tumor), and pain (dolor). Theheat and redness results from dilatation of arterioles,capillaries, and venules with increased permeability andblood flow; the swelling is formed by the accumulationof fluid, plasma proteins, and leukocytes which migrateinto the site of infection; and the pain is caused by tissuetension from the edema.

Inflammation may be caused by a local protectivereaction of vascularized tissue to injury resulting fromeither a noxious or innocuous stimulus. The mainfunction of the inflammatory response is to destroy,dilute, or “wall off (sequester) both the injurious agentand the injured area. Noninfected inflammation is ●normally associated with the same series of responses(i.e., local heat, redness, swelling, and pain) as

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infection. In fact, the most important cause ofinflammation is bacterial infection, although othertypes of noxious injuries, such as the death of tissueand thermal, chemical, or physical trauma may alsoinduce a similar pattern of inflammatory reaction.Rheumatic and allergic diseases are caused byinnocuous stimulus in which the inflammatory responseitself is the cause of tissue damage. The exact details

of cellular cytosine radiation of the inflammatory

response are beyond the scope of this continuing

education (CE) lesson.

Modalitia for Localization of Infection and

Inflammation

Computed tomography (CT), ultrasound, magnetic

resonance imaging (MRI), and radionuclide

scintigraphy have all been used in the workup of

patients with suspected infectious and inflammatory

disorders. CT has the advantage of precise anatomical

localization of abscess and is not hindered by tubes,

sites of drains, or the presence of open wounds. CT is

a relatively fast procedure that can be performed with

a minimum of patient discomfort. However, false

positive diagnosis of abscess formation may be seen in

tumor necrosis, thick-walled cysts, and unpacified

bowel loops. In addition, if there are no localizing

signs or symptoms present, a whole-body CT scan is

time-consuming and results in a high radiation dose to

the patient.

Ultrasound has some advantages over CT, MRI,

and radionuclide scintigraphy as it is the fastest and

least expensive mode of abscess imaging. It also has

the advantage of imaging in multiple planes, whichmay allow more accurate lesion localization.However, ultrasound evaluation is highly dependentupon the operator’s expertise, and its ability to obtainimages may be limited when open wounds, drain lines,or tubes exist. Gas and bone also interfere with theperformance of ultrasound. It also demonstrates lowspecificity since some conditions, such as hematoma,lymphocele, cyst, seroma, and fluid filled bowel loop,may mimic the sonographic appearance of an abscess.

MRI is a new modality that is also useful in theworkup of a patient with infection/inflatnmation. It hassome of the advantages of CT as it is not affected byopen wounds or drains. Like ultrasound, MRI canimage in multiple planes. Like all the imagingmodalities, MRI also has limitations as to thespecificity of a positive finding when any cause ofincreased intercellular fluid (edema) may mimicinfection/inflammation. It is also costly and very timeconsuming to do a whole body MRI study.

Both CT and ultrasound require the formation ofabscess for accurate diagnosis, whereas radionuclidescans and MRI can detect the site of

infection/inflammation even prior to abscess formation.me major advantage of radionuclide imaging is itsability to survey the entire body with a singltexamination.

The question of which modality to use for theevaluation of infection and when to use it can be besianswered by examining the clinical condition of thepatient. Patients who are not critically ill or who haveno localizing signs should be studied first with a

radionuclide scintigraphy study (e.g., lliIn-labeledleukocytes). If, however, patients require promplintervention or have localizing signs, CT, ultrasound, orMRI should be used initially.

Many radiopharmaceuticals have been used in theevaluation of patients with suspected infectious and/o~inflammatory process. Before the advent of CTscanning, brain scintigraphy was used in the evaluationof brain abscess and cerebral inflammation (l-4).

Liver/spleen scintigraphy was used prior to the days oiultrasound and CT in the evaluation of hepatic abscess(5,6). Currently, bone scintigraphy remains a veryimportant part of the diagnostic armamentarium in theevaluation of musculoskeletal sepsis (7). Upon reviewof data compiled horn a series of articles, bone scanningshows a sensitivity of 94% and a specificity of 95% inbone that has not been complicated by previousinfections, trauma, or surgery. However, the specificityof bone scanning drops significant] y to a range of 33%when the disorder is complicated by remodeled bone (7).In the case of bone remodeling, additional imaging withradiolabeled white blood cells (WBCS) or ‘7Ga-citrate isnecessary for further delineation. The remainder of thisCE lesson will be limited to the radiopharmaceuticalswhose primary role involves the general evaluation ofinfection and inflammation,

Ideal Characteristics of Radiopharmaceuticals forImaging of Infectious and Inflammatory hions

The value of radionuclide scintigraphy is wellestablish. The following represents a “wish list” forthe characteristics of an ideal radiopharmaceutical inlocalizing infectious and/or inflammatory lesions:

1. High sensitivity and specificity in differentdisease sites to help ensure that an accuratediagnosis can be made. Biological properties ofthe imaging radiopharmaceutical, such as rapidlocalization in the focus, a high target-to-nontarget ratio, and rapid blood and backgroundclearance, are all critical in contributing to highsensitivity. The agent should localize only insites of infection/inflammation for idealspecificity.

2. Easy availability and simple preparation.

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3. Inexpensive cost.

4. Acceptable radiation dosimetry without toxicityso that serial studies can be performed whenneeded.

Radiopharmaceutiub for the Detection of Infectiousand Inflammatory hions

A number of radiophmmaceuticals are available fordiagnosing infection and inflammation (Table 1). Eachradiopharmaceutical for use in imaging infectious andinflammatory lesions (listed in Table 1) will bediscussed in detail in respective sections which follow:

Radionuclid=● ‘Ga-citrate● ‘1‘hc13

RadiolaheledAntibodies● 1*lIn-labeled nonspecific polyclonal

human immunoglobulin-G● -c-labeled rnonoclonal

antigranulocyte antibody

Other Agents9 ~c-labelti nanocolloids● l*’In-labeled avidin-biotin

RADIONUCLID~

‘Ga-CitrateGallium-67 citrate (c7Ga-citrate) (Figure 1) was first

demonstrated to accumulate in inflammatory lesions in1971 (8), and since then this radiopharmaceutical hasbeen used routinely for the detection of infection andinflammation. ‘7Gahas the advantage of not requiringany special preparation before administration.However, after two decades of clinical use, themechanism of ‘7Ga localization in abscess and otherinflammatory sites is not yet fully elucidated.Localization of ‘7Ga has been attributed to [a] bindingto intracellular lactoferrin of leukocytes (9,10), @]direct binding to microorganisms (10-12), and [c]abnormal passive diffusion of the b7Ga-transferrincomplex through the alterd vascular endothelium ofinflamed tissue (13).

H2C–COO”\\

I\L,

HO– C–COO- ----~/0’

//H2C–COO-0’

Fiure 1. Chemicalstructure of ‘Ga<itrate.

Although ‘7Ga has been shown to be useful in theevaluation of patients with infectious and inflammatorylesions, ‘7Ga has a number of disadvantages. me highradiation absorbed dose to the patient limits themaximum amount of ‘7Ga which can be administered.The physiological localization of ‘7Ga in the liver and ifinormal renal and bowel excretion may obscure lesionsand complicate the diagnosis of abdominal abscess.Even with bowel cleansing prior to imaging, activity inthe gut is usually seen on the b7Ga scan and oftennecessitates delayed imaging (14). The lack ofspecificity of b7Ga uptake is especially a problem with

cancer patients, since ‘7Ga is taken up by a variety oftumors. In addition, areas of recent surgery andnormally healing wounds are visualized with ‘7Ga;

therefore, 67Ga is less useful in the postoperative periodwhen detection or exclusion of an abscess may be

extremely important. All of these reasons make ‘7Ga-citrate a less than optimal radiopharmaceutical fordelineating infectious and inflammatory lesions.However, there are areas where ‘7Ga is the agent of

choice. When a patient presents with a fever ofunknown origin (FUO), the cause may be infection,

ma] ignancy, or other chronic inflammatory or

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granulomatous process. Nonspecific ‘7Galocalization inthese types of lesions can actually be a benefit in thiscircumstance since detection and localization is moreimportant than a specific diagnosis. Gallium is alsobetter than other agents (i.e., ‘llIn-labeled leukocytes)for the diagnosis of disc space infection (15,16) and forinfection foci of more than two-weeks duration (17).

lllInCl~Iridium-l 11 chloride (*llInCl~)was first suggested in

1976 for use in patients with inflammatory disease (18).Sayle et al. (19) have demonstrated its potential ousefulness in the detection of suspected abscess.However, the accumulation of lllInCl~ activity in thekidneys and bladder makes the interpretation of

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infectious and inflammatory disedse at these sitesdifficult. As with ‘i7Ga, *“InCl, also localizes inpatients with tumor, and this may be misinterpreted as

●acute abscess. However, unlike ‘i7Ga, ‘llInCl~ is notexcreted through the. gastrointestinal tract. This isparticularly advantagwus in patients with abdominalinflammatory disedse who cannot undergo an adequatebowel cleansing procedure. Due to the limited clinicalexperience with IllInC]~ in detection of infectious andinflammatory lesions, more extensive investigation andconfirmation of its clinical usefulness is needed in thisapplication. “’InClq has been approved recently by theU.S. Food and Drug Administration (FDA); however,its indication for usage as stated in the package insert(20) is for radiolabeling OncoScint” preparations usedfor the detection and staging of colorectal cancer (21).

RADIOLABELED LEUKOCYTES

Biological Functions of bukocyt~WBCS (leukocytes) play a major role in the body’s

defense mechanism in which they immobilize,phagocytize, and kill organisms involved in infections,The usual range of leukocytes in the normal adult is5,000 to 10,000 WBCs/mm3. These WBCS includegranulocytes (50% to 83%), monocytes (O% to 7%),

and Iymphocytes (20% to 40%). The granulocytes aremade up ()f three subclass es--neutrophils[polymorphonuclear leukocytes (PMNs)], basophils,and wsinophils, whose names are derived from theirstaining characteristics. PMNs, which phagocytize anddegrade many types of particles, serve as the body’s firstline of defense when tissue is damaged or foreignmaterial gains entry. PMNs congregate at sites inresponse to a specific stimulus through a process knownas chemotaxis. The function of basophils in thecirculation is not clearly understood; msinophils andlymphocytes are classically msociated with immunereaction, and lymphocytes are involved with chronicinfection. Monocytes are phagocytic cells and serve asa secondary defense mechanism.

Since leukocytes comprise the primary defensemechanism against infection, researchers haveattempted to image abscesses and other sites of infectionby labeling these cells with a variety of gamma-emitting radionuclides. The use of radiolabeled cellshas certain advantages when compared withconventional radiopharmaceuticals; first, the normal“homing” functions of cells are exploitd to carry theradiopharmaceuticals to specific sites in the body and,second, it allows tie biodistribution of normal anddiseased blood cells to be studied noninvasively.

@ Collection RBC sedimentation

&’\-- 1 of blood

❑●*h

&

n

- \/1. Transfer LRPRP

v,

Reinfection

$ f

@’@ ] @’@

Cell labeling

—F~ure 2. Basic schematic illustration depkting the various steps kvolvd h the radiotabeting of leukoc~. Please refer

h text for detaited description. LRPRP: leuk~yterich platelet-rich plasma, PRR ptatela-rich plasma, PPP:platelet-tn30r ptasma.

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General Steps for Radiolabeling LeukocytesSince most of the radioactive chelating agents used

for leukocyte labeling are nonselective (i.e., theytag all cells), it is necessary to isolate the leukocytesfrom the remainder of the cellular components ofblood (erythrocytes, 1ymphocytes, monocytes, andplatelets) before radiolabeling can be performed. Thegeneral steps irtvolvd in the radiolabeling ofleukocfies are described above in Figure 2.

Collection of Blood. In order to obtain a su~cientlylarge population of leukocytes. approximately 50-100MI of venous blood is withdrawn from the patient.However, the requirement for a large volume of bloodmay present problems with pediatric patients. For thisreason, the Childrens Hospital of Los Angeles. LosAngeles, California, has established a guideline inwhich the volume of blood withdrawn for aradiolabeled leukocyte study is 1 ml/kg for a pediatricpatient age 2 to 18 (22). The minimum and maximumvolumes of withdrawn blood are 20 ml and 50 ml,respectively (22). Another guideline for blood volumeto be withdrawn in children is 15-30 ml for bodyweights less than 100 lb, and 30-60 ml for bodyweights greater than 100 lb (23).

Both acid citrate dextrose (ACD) and heparin havebeen used as an anticoagulant with the collection ofpatient blood; however, there is a consensus that ACDis a better anticoagulant than heparin for this purposebecause leukocytes show less tendency to adhere toplasticware and to each other with ACD (24,25),However, Peters et al. (26) have found that ACDpromotes microaggregation during labeling.

Separation of “Crude”Uukocytes. To accelerate therate of sedimentation of the erythrocytes,polysacchaide (e.g., dextrose, methylcellulose, orhydroxyethyl starch) is added to the anticoagulatedblood. These agents speed the sedimentation processby causing the red blood cells (RBCS) to clumptogether, thereby reducing the required sedimentationtime by one-half. Hydroxyethyl starch (HES) [6%hetastarch in 0.9% NaCl solution (Volex@, AmericanCritical Care, McGaw Park, IL; Hespan@, Du PentPharmaceuticals, Wilmington, DE)] is an effectivesettling agent when used one pafi HES to five partsblood (24). Roy et al. (27) have shown that HESincreases the RBC sedimentation rate and the leukocyterecovery is greater than with dextrose. In addition,HES is an FDA-approved drug and is not associatedwith allergic reaction (28), whereas dextrose andmethylcellulose have been reported to produce adversereaction in some patients. Special care is required forstorage as the 6% HES is available in 500 mlcontainers only and does not contain any bacteriostatic

agent. Since HES readily supports growth of organisms,the commercially available 6% HES solution is,therefore, designed for single use only.

Following approximately 30 to 45 minutes of gravitysedimentation, the supernatant is rich in leukocytes o(approximately 70%) and platelets, and is known asleukocyte-rich platelet-rich plasma (LRPRP). However,one to two red cells per leukocyte as well as the majorityof platelets are still present in the supernatant (29). Toeliminate RBC contamination of the LRPRP, hypotonicsaline or ammonium chloride (29,30) has been used tolyse RBC. However, decreased circulation half-timesand increased hepatic uptake of leukocytes have beenfound following the hypertonic or hypotonic RBC lysis,possibly due to WBC damage (31). In our institution,we use a centrifugation method (30 g for five minutes orILRPRP layer) (Figure 2, sub-step 2) to remove anyremaining RBCS contained in the plasma.

To remove the smaller and lighter platelets in theLRPRP layer, the supernatant is carefully separated fromthe RBC pellet and is then centrifuged at 100 g for 8minutes to obtain the leukocyte pellet (Figure 2, sub-step3). A “rocking” step has been developed by Kaminsky(32) to be used in order to deplete RBC contamination(if this has not already been accomplished by othermeans). Following isolation of the leukocyte button, theleukocytes are resuspended in 4 ml of normal saline andthen allowed to rock upon a specially modified rockerarm for 15 minutes (32). Kaminsky has demonstrated o

that this patented method results in a 1% to 5% residualRBC contamination as compared to 20% contaminationof tagged RBC without the RBC depletion steps (32).

The platelet-rich plmma ~RP) is drawn off andtransferred to a fresh centrifuge tube. This tube is thencentrifuged at 3,200 g for 10 minutes to form platelet-poor plasma (PPP) (Figure 2). Following the removalof the PRP, the leukocyte pellet is resuspended inACD/saline solution and labeled by the addition of aradioactive completing agent (Figure 2).

Separ~”on of “Pure” Gmn40cytes. When “pure”granulocyte preparations are requested, granulocytescannot be separated from monocytes and lymphocytes bydifferential centrifugation. However, because of slightdifferences in cell density, they can be separated by thedensity gradient centrifugation method. In centrifuging;ith discontinuous density gradients, the cells migrateuntil they reach the surface of a solution whose densityis equal to or greater than their own; hence, cells ofvarying densities come to rest at different depths. Byusing a multiple-density solution (e.g., the widely-usedFicoll-Hypaque discontinuous density gradient), ●granulocytes, monocytes, and lymphocytes can beseparated with less than 10% cross-contamination(24,33) (Figure 3). Other discontinuous density

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gradients that have been used include Percoll (34) andMetrizamide (35). Although Ficoll-Hypaque gradients

, have been widely used for separation of neutrophils,

o

some studies have shown that this mixture may have anadverse effect upon leukocyte viability (36). Percollhas been recommended as a more innocuous substitute(24,36) since Percoll is a colloidal silica coated withpyrophosphate which renders it nontoxic. Leukocytesseparated by the use of Percoll gradients have beenshown to have normal Chemotidxis and bactericidalactivity (36,37). Other cell-separation techniques thathave been used for obtaining pure granulocytesinclude column separation (37,38), phagocytosis(29,39), centrifugal elutination (24), and flowcytometry (24,40).

Pure Granulocytes vs. Crude Leukocytes.Schauwecker et al. (41) have conducted a study tocompare the advantages and disadvantages of purifiedIllIn-labeled granulocytes with lllIn-labeled mixedleukocytes, They have found that the crude leukocytepreparation is probably the agent of choice for imagingof infection because:

1. cell separation and labeling of a standardmixed cell preparation is easier.

o2. in acute infection, the uptake sensitivity of

either pure granulocytes or mixed cells is highand not statistical y different.

3. mixed cell preparations have a slightly superiorsensitivity when compared with pure

Ficoll-Hypaque

gradient II

(1.12 g/mL) v

granulocytes for chronic infections; this is likely due tothe presence of labeled lymphocytes.

In addition, Peters (42) has demonstrated that, in hisexperience, pure granulocytes are markedly activated invivo and show prolongd retention in the lung.However, pure granulocytes are useful in certain specificcircumstances:

1. When patients are without neutrophilia.

2. When granulocyte deposition may be obscuredagainst a high blood background resulting fromRBC and platelet-associated activity as incardiovascular inflammation when mixed cellsare used (42).

3. When granulocyte quantitation or kineticinformation is required.

Cell Lubeling. Cell labeling is a very simple and rapidprocedure involving the addition of the radioactive agent(e.g., l’lIn-oxine, *’lIn-tropolone, ‘Tc-sulfur colloid,*Tc hexamethylpropyleneamineoxime ~Tc-HMPAO])to the leukocytes and incubation for five to fifteenminutes at room temperature or 37 “C.

It is desirable to incubate the leukocytes in plasma inorder to maintain cell viability. The granulocytes label-ed in plasma clear rapidly from normal lungs, migrate tosites of abscess more rapidly, and demonstrate a reducedquantity of radioactivity absorbed by the liver (43).However, certain ligands such as oxine have less affinityfor lilIn than plasma protein; therefore, leukocytes mustbe removed from the plasma for labeling.

01,650 g

@

Plasma

Mortocytes +

lymphocytes

platelets

Granulocytes

+

Erythrocytes

Feure 3. Wierent cell components of Ieukwytes separated by using two Fwoll-Hypaque discontinuous density gradienfi.

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Factors Which Aff=t Labeling EfficiencyThe ability of a radioactive ligand to label

leukocytes is usually assessed by measuring thelabeling efficiency (LE). However, it is often difficultto compare values quoted in the literature since thelabeling conditions in different studies me rarely thesame.

The LE of radiolabeld leukocytes is affected by thefollowing factors:

1. pH of the mtiium. Cells are usually labeledat a pH between 6.5 and 7.5 to maintain theviability of the cells.

2. Concentration of ligand. Approximately 101to I@ times more ligand than l]lIn is requiredbefore cell labeling will occur. Withphysiological saline, the maximum LE isobtained over a much wider concentrationrange than in plasma. Thus, it is veryimportantto use the optimum concentrationofIigand, especially when labeling cells inplasma.

3. Cell concentration. In general, the LEincreasw with cell concentration, althoughhigher cell concentrations are needed whenlabeling cells in plasma (44-47). This is due toa portion of the radionuclide (especially lllIn)being diverted to plasma components.

4. Incubation time and temperature. Celllabeling is usually rapid, reaching a plateauafter 5 to 30 minutes incubation at 25 ‘C or37 “C (44,48,49). However, a much slowerrateis observed when the labeling is performedat 4°C (46,50).

Quality Control of Radiolabeled ~ukocyt~Ideally, one wants to obtain a high LE; however, it

is much more importantto maintain the viability andfunction of the labeled leukocytes. Certain degrees oflabeling injury to the leukocytes may occur during invitro manipulation; therefore, it is essential to performquality control on radiolabeled leukocytw to ensurethat viability and function of the labeld granulocytesare well presemed. Cell viability may be assessedeither in vitro or in vivo.

In Vtiro Tests. Various in vitro methods have beenused; these include quality control Ficoll-H ypaquedistribution analysis which shows the percentage ofradioactivity in each cellular component (51) (Figure3), trypan blue exclusion test which evaluates celldeath (52), dual-filter radiochernotaxis assay (53),

.

phagocytosis and intracellular killing capacity ofgranulocytes (54), and others. However, the in vitro ,tests are not practical on a routine basis as they are time-consuming and have failed to show damage which isclearly seen in vivo (55). ●In Vivo Tests. The real test of the integrity of thelabeled cells is upon reinfection into the patient. Cellrecovery, as measured by the percentage of radiolabeledWBCS circulating in the blood after reinfection anddisappearance half-time of labeled leukocytes, givessome measurement of in vivo behavior of theradiolabeled granulocytes. The leukocytes with lowerviability are irreversibley sequestered in the liver,whereas samples contaminated with RBCS andlymphocytes result in greater splenic activity (35,36). Inaddition, damagd granulocytes are reversibly retainedin the lung (35). Datz et al. (57), however, could not ‘find my significant correlation between the earlytransient lung uptake of radiolabeled leukocytes and thedecrease in sensitivity for detecting infection at earlyimaging times (< 6 hours). Weisberger et al. (54) havesuggested that the early lung localization may representonly the natural physiologic marination of PMNleukocytes in the lung capillaries.

Considering the drawbacks associated with the qualitycontrol methods mentioned above, it has been suggestedthat routine complete blood count (CBC), white celldifferential count, microscopic examination, and o

determination of LE are adequate to maintain qualitycontrol of radiolabeled leukocytes (25).

tiukocyte Couti. In order to obtain adequate imaging,approximately 1 x 10s leukocytes are required whenautologous granulocytes are used (59). For reinfection,when non-autologous cells must be used, McCullough etal. (60) have suggested that no fewer than 2-4 x 108cells must be given since histocompatibility factorspresent in some patients may rduce the intravascularsurvival of the non-autologous cells.

lllIn-labeled Leukocyt=In 1976, McAfee and Thakur (29,39) demonstrated

that lllIn oxine could be used to label leukocytes. Theneed for a radiopharmaceutical such as lllIn labeledleukocytes can probably be best measured by the morethan 700 investigational new drug applications,sponsorti by the Amersham Corporation, on file withthe FDA, prior to official approval of ‘llIn oxine onOctober 17, 1985 (61).

Although *Tc-oxine was also investigated and foundto be useful, l“In was preferred to *Tc because its ●longer physical half-life (67.2 hours) allowed imaging at24 hours following administration of the labeled WBCSwhen blood and background activities were low.

8

Repeated investigations up to one week post-injectionare also possible with ‘llIn. *l~Inhas a high abundance

*of two gamma photons at 171 keV (90% abundance)

o

and 245 keV (94% abundance) which are suitable fornuclear medicine imaging. However, since lllIn is acyclotron-producd radionuclide, it is relativel y expens-ive and not readily available. In addition, because ofhigh radiation dosirnetry, especially to the spleen, thedose and resultant count activities are limited.

m/\\’N/OH

o

0Oxine

n

/

/ \OH\ ~~

N— I

o

Tropolone 2-Mercaptopyridine-N-oxide

o Fgure 4. Chemical structurm of the Iiiands used toform iridium complexes for leukocytelabeling.

Mechanism of Leukocyte Weling with lllIn-Oxine.Oxine (8-hydroxyquinoline) is a lipophilic chelatingagent that has been usd as a topical bacteriostatic andfungistatic solution, antiperspirant, and spermicide(Figure 4). Iridium forms a saturated (1:3) complex(Figure 5) (theprecise chemical structure of ‘~*In-oxine

Figure 5. Possible chemical structure con~-uration ofindium-liiand complexti.

is unclear at this time) with oxine that is neutral andlipid-soluble. This complex is able to penetrate the cellmembrane of leukocytes. Once within the cell, the lllln-oxine complex dissociates, and the lllIn binds to nuclearand cytoplasmic proteins which have higher bindingaffinities for the iridium (44) (Figure 6). Some of thereleased oxine readily diffuses from the cells, and somemay chelate with other cell ions (62).

Biological Distribution of ‘llIn-Oxine-Labeledbukocytes. Usually in a mixed-cell population, withthe cell pellet containing approximately 34 x I@ WBCSafter following the recommended cell labeling procedure,the cell-bound activity distribution in neutrophils,1ymphoc~es, and RBCS is 80%, 15%, and 5%,respective y, whereas pure granulocyte preparationsobtained by Ficoll-Hypaque separation have 98%radioactivity associated with neutrophils (63,64).Goodwin et al. (65) have achieved art LE of 84% &9%,

WBC

111l“ (Oxine)

F~ure 6. %hematic diagram of the proposed m=hanism for leukocyte labeling with lllIn+xine.

9

and in patients with WBC counts above 15,000 mm3in peripheral blood, it was usually > 90%.

The package insert for Iridium In 111 OxyquinolineSolution (66) re~mmends that the maximum timebetween drawing patient blood and reinfection of ~l~In-labeled leukocytes should not exc~ five hours. It isalso recommended that the labeled WBCS should bereinjectd within one to three hours after theradiolabeling procedure (66,59).

Irnmediately after reinfection of the labeledleukocyt~, activity is distributed throughout the lung.One-half of this lung activity clears within 15 minutes,and it is no longer present by four hours (56). Activityis distributed subsequent y to the spleen (30%), liver(30%), and bone marrow (35%) (66). Theconcentration in the liver and spleen remainsunchanged for up to 72 hours (66). Accumulation ofapproximately 60% of the injected dose in the liver andspleen appears to represent uptake of viable cells aswell as removal of damaged cells or cell fragments.The radioactivity found in bone marrow may representtransferrin-free ~~lln carrier or migration of cellularcomponents. Between 9.5 % to 24.4% of the injecteddose remains in whole blood and clears with adisappearance half-time of 5 to 7.5 hours (65,56,67).The recovery of ‘*]In-labeled leukocytes has beenmeasured to be 50% to 75% using a mixed leukocytesuspension which was contaminated with RBCS(44,56). Release of radioactivity from the labeled cellsis about 3% at one hour and 24% at 24 hours (66).

Dosage. The standard adult (70 kg) dose for “lInlabeled leukocytes is 18.5 MBq (500 pCi), whereas thepediatric dose can be adjusted by using a standard table(68) based upon the Vapower of a child’s body weightwith a minimum dose of -3.7 MBq (- 100 pCi) assuggested by Marcus et al. (69)

Imu@”ng Protocol. The localization of cells atinfectious and inflammatory sites is not usuallyapparent during the first hour post-injection but may bevisible at four hours. The relative accumulation ofradioactivity in inflammatory lesions reaches amaximum at 24 hours which also coincides withavailable activity considering a blood disappearancehalf-time of 5 to 7 hours. Images obtained at 48 hoursmay be beneficial to monitor intestinal activity seenearlier. If the bowel activity persists at 48 hours, thisis an indication of infections and/or inflammatorylesion sites. If, on the other hand, this intestinalactivity disappears at 48 hours, this would then indicatethat the radioactivity originated from swallowedleukocytes caused by nasopharyngeal or pulmonaryinfection rather than inflammatory lesion. In normalvolunteers, only negligible amounts of “lIn (less than

1%) are lost from the body in urine or feces. Therefore,the elimination of injectd ‘llIn from the body isprobably through physical decay to stable cadmium (66).

lllIn and Other Chehzting Complexes. Other chelatingagents of iridium have been studied for their ability tolabel cell components. A major reason for pursuing theuse of other agents has been to achieve the labeling ofleukocytes in plasma in order to preserve WBC viabilityand function. Two chelating agents that have beenused for leukocyte labeling in plasma are tropolone(2-hydroxy-2,4,6 -cycloheptamine-l -one) andmercaptopyridine-N-oxide (MERC) (Figure 4). Bothtropolone and MERC are lipophilic chelating agentsthat label leukocytes using the same mechanisms as *llIn-oxine. During a four-month period in late 1981 andearly 1982, three independent reports appeared in theliterature on the use of lllIn-tropolone for labeling bloodcells (70,71 ,34). MERC was first reportd by Thakurand Barry in 1982 (49).

Both tropolone and MERC are water-solublebidentate chelating agents which can be complexd with*~~Inand will label blood cells in plasma. This seems tobe an advantage when compared to oxine, whichrequires the removal of plasma during the labelingprocess and, therefore, subjects the cells to possiblephysical or physiological impairment. However, resultsof studies direct]y comparing the in vitro and in vivobehavior of leukocytes labeled with oxine, tropolone, orMERC are confusing. Some investigators havediscovered that tropolone used in the labeling processmay diminish chemotactic properties of the labeledleukocytes (72), and that the tropolone may be moretoxic to the cell than oxine (73). Although “’ln-tropolone-labeled leukocytes have proved slightl y moresensitive than leukocytes labeled with *l]In-oxine for thedetection of abscess on images taken one to four hourspost-injection, the difference in lesion detectability is notstatistically significant between the two agents at 24hours. Goedemans et al. (74) have conducted a studycomparing the LE among oxine, tropolone, and MERC.They found that if the plasma residue is greater than30%, neither “]In-tropolone nor ‘l’In-MERC exceeds theLE of lllIn-oxine. This seems to suggest that neithertropolone nor MERC is compietel y free from the“plasma competition” effect with respect to cell LE. Inanimal studies (37,49), lllIn-MERC has been shown tohave less uptake in muscle, liver, and spleen at 24hours, suggesting that leukocyte function may be betterpreserved with this chelating agent. However, theresults from a clinical comparison study (72) indicatethat no significant difference in sensitivity was foundbetween 11’In-oxine-labeledleukocytes and ~llIn-MERC-labeled leukocytes.

The conflicting results mentioned above may be the

10

main reason that there is not yet a manufacturer who iswilling to initiate clinical trials for commercializationof either tropolone or MERC. Currently, liiIn-oxineis the only chelating radiopharmaceutical approved inthe United States for leukocyte labeling.

~c-Labeled Leukocyt~bukocytes Lubeled with ‘Tc Using Phagocytic

aruf Pretinning Methods. Prior to 1976, blood cellswere labeled with one of three gamma-emittingradionuclides: 51Cr, b7Ga, or *Tc. Neither 51Cr nor‘7Gahas ideal physical characteristics for imaging. Incontrast, ‘iTc has excellent physical properties, andprevious attempts have been made to label leukocyteswith *Tc by either phagocytic uptake of various‘Tc-sulfur colloid preparations (75-80) or byincubation of *TcO~ with pretinned leukocytes(similar to the classic method used for RBC labeling)(81-84).

In theory, neither the phagocytic nor the pretinningmethod for leukocyte labeling requires cell separation.However, in practice, separation techniques are stillperformed due to the poor LEs in vivo and in rton-separated cells. Drawbacks with these two methodscontinue to be poor LE, difficulties in completeseparation of *Tc products from the *Tc-WBCS,significant activity rinsed from the leukocytes duringthe labeling process, and rapid elution of radioactivityfrom the cells.

The mechanism for phagocytic labeling appears tobe surface adherence rather than phagocytosis, and isthe cause for lung and liver *Tc uptake followingrelease of the colloidal products (75-80,85). UsingWBCS labeled with ‘Tc colloids, Hanna et al. (86)have found that prolongd lung transit time can beexplained by aggregation or activation of theneutrophils. Although interest in *Tc-colloid hasrecently been revived by demonstration that, when theparticle size of the colloid is rigidly maintained at 2.1pm, the technique does work (87,88). Many of thecommercial sulfur colloid kits contain colloid in whichthe majority of the particles are between 0.1 and 1 ~m,with only 5% greater than 1 pm (89). Marcus et al.(90) have reported success using ‘mTc-microaggregatedalbumin colloid-labeled WBCS in acute infection.Acute infection was visualized by 30 rein, whilechronic or low-level infection and/or inflammation tookseveral hours to identify (90). Pending further clinicaltesting by other investigators, this method promisessubstantial clinical usefulness.

The pretinning method for labeling *Tc-leukocyteswith different agents has been previously investigated(81,82), and the glucoheptonate method appears to bethe best, demonstrating LEs of 48.3%+ 12.6%. Arecent report indicates that consistent labeling yields of

up to 90% have been achieved. However, in vivorecovery and biodistribution of *Tc-WBCS (labeledusing the stannous glucoheptonate pretiming method) indogs were found to be low and variable unless bufferedsaline was used (91). Unfortunately, the use of bufferedsaline in the glucoheptonate method reduces the LE to50% to 60% (91).

9%Tc-HMPA@Labeled Leukocytes. In 1986, Peters etal. (92) described the possibility for labeling leukocytwwith *Tc-HMPAO (Figure 7), with LEs ringing from41% (93) to greater than 86% (94). This newradiopharmaceutical (i.e., *Tc-HMPAO not labeled toWBCS) has been introduced for cerebral perfusionimaging. Due to its lipophilic character, like l]lIn-oxine,this agent is able to diffuse through the cell membraneand is rapidly incorporated into the leukocytes to bindintracellular] y. However, the application of *Tc-HMPAO in labeling WBCS as an aid in detectinginfectiouslinflammatory lesions is not listed in themanufacturer’s package insert (95).

Compared with lliIn-labeled leukocytes, *Tc-HMPAO-labeled leukocytes offers all of the potentialadvantages of a ‘iTc agent, such as convenience, betterimaging characteristics, and reduced patient radiationabsorbed dose.

The cost-effective advantage, which is a benefittypical of using ‘Tc agents, is negated by the expenseof the HMPAO cold kit. The concept for fractionatingthe HMPAO kit has been proposed recently (96-100) inorder to utilize HMPAO in a more cost-effectivemanner. Other constraints imposed by the use of *Tc-HMPAO include the very short shelf life of *Tc-HMPAO (i.e., 30 minutes post-reconstitution) (95) andcertain restrictions for the *Tc-pertechnetate that mustbe used in the preparation of *Tc-HMPAO (95).

The use of *Tc-HMPAO to label leukocytes hasresulted in a substantial reduction in radiation exposureto patients (e.g., approximately 30% reduction ofradiation absorbed doses to liver, spleen, and bonemarrow) with 185 MBq (5 mCi) *Tc-labeled leukocytescompared to 18.5 MBq (0.5 mCi) “lIn-labeledleukocytes (101). However, there is a higher radiationdose to the person who performs the leukocyte radio-labeling procedure with the *Tc agent. Ponto et al.(102) have demonstrated that personnel doses from the*Tc-HMPAO leukocyte labeling procedures (startingactivities: 518-588.3 MBq [14.0-15,9 mCi]) are 3.3times higher for the whole body and 4.6 times higher forthe hands than are the doses from l]lIn leukocyte labelingprocedures (starting activities: 24,1 to 25.9 MBq [0.65-0.70 mCi]). Therefore, it is important for nuclearpharmacists and nuclear medicine technologists toimprove upon methods usd for radiation protection inthe labeling process of ‘Tc-HMPAO leukocytes.

11

Biodistributionof *Tc-HMPAO-LaNed h UkocvtaThe initial biodistribution of ‘Tc-HMPAO-labeledleukocyt= is similar to that with “’In-labeledgranulocytes, showing a rapid initial lung transit,prominent splenic activity, bone marrow activity, andhepatic activity. The blood disappearance curves andpercent recovery of *Tc and ‘~]Inare also comparable(103,104). However, in the studies done by Mock etal. utilizing leukocytes labeled with ‘Tc-HMPAO andlliIn-tropolone (105), they have discoverd that therespective blood clearmce half-times and percentrecovery of the two agents were often comparable, butin some cases, the percent recovery of *Tc in theblood was lower. This was probably due to differentradiochemicalpurityof the ‘Tc-HMPAO preparationsfrom batch to batch.

Udike ‘i*In, ‘Tc activity is also seen in urine,occasionally in the gallbladder, and from about fourhours, consistently in the bowel.

~. Costa et al.(106) studied 47 patients without known inflammatorybowel disease (IBD) or abdominal infection, and

labeled cells (which severely limits quantification andkinetic studies) may be the reason for the higher renal,biliary, and bowel excretion. The nonspecific bowelactivity (including biliary activity) and urinary activity isprobably due to a secondary hydrophilic *Tc complex,and ordy to a small extent to the primary lipophiliccomplex or free [%Tc] pertechnetate, since nosignificant brain, thyroid, or stomach uptake isdemonstrated on either the four hour or 24 hour images.The major chemical composition of spontanaus elutionin vitro has also been confirmed to be a secondarycomplex of *Tc-HMPAO (51.8% to 65.2%) (109).

The unbound *Tc activity which localizes in thekidney and bladder may make the detection of urinarytract infection difficult. The nonspecific bowel activityassociated with *Tc-HMPAO-labeled leukocytes mayalso present difficulties in the detection of abdominalinfection. Surgical management of acute intrabdominalsepsis requires that accurate localization be made asquickly as possible. It has been reported that fourhours is the earliest time at which positive uptake inintraabdominal sepsis is routinely noted (92,110) in spiteof the nonspecific bowel activity. A recent report by

OH HO

~,1- HMPAO

H3C ~H3

A

.“

0, 0H“

99mTc.d,/-HMPAo

Fwure 7. Schematic repr=entatinn of chemical structures for d,I-HMPAO and ‘Tcd,l-HMPAO.

found that activity in the small bowel at 30 to 60minutes post injection was seen in 16% of the *Tc-labeled WBC md in 7% of the ‘**In-labeled WBCstudies. This percentage increased to 95% of the‘Tc-labeled WBC and 33% of the’l*In-labeled WBCstudies at 18 to 24 hours post injection. In anotherstudy (107), the urinary bladder was consistent] y seenon the 30 to 60 minute study, with approximate y 17%cumulative urine activity at 24 hours with ‘Tc-labeledWBC. This was not the case with the 11’Instudies. Ithas been found that the in vitro elution in saline fromWBCS of liiIn ad *Tc was 2% and 9% per hour,respective y (104,108). The higher elution from ‘Tc-

9

i

●

Lantto et al. (111) showed sensitivities for abdominalinfection of 74%, 88%, 95% and 96% at two minutes,30 minutes, two hours, and four hours, respective y.Allan et al. (112) have demonstrated that one-hour *Tc-HMPAO leukocyte images for IBD are as informative asthree-hour lllIn-oxine labeled leukocyte images. Thismay be related to better resolution associatd with thehigher photon flux and optimal imaging energy of *Tc.The improved resolution is probably also due to theshorter imaging times, which minimize bowel motion oand probably also minimize intraluminal movement ofactivity.

12

Selwtive Radiolabeling. Peters et al. (107) havesuggested that a pure radiolabeled granulocytepreparation, without the necessity for granulocytepurification from a mixed leukocyte preparation, canbe achieved with ‘Tc-HMPAO. Their suggestion isbasal upon two distinctive features of *Tc-HMPAO.First, leukocytes can be labeled in the presence ofplasma proteins by using *Tc-HMPAO (92); second,radioactivity elution occurs more rapidly from othercell types than from granulocytes (92,108) in a *Tc-HMPAO-labeled leukocyte preparation. However,Kelbaek et al. (113) argue that since Peters et al. didnot associate their findings with the cell differentialcounts in their final suspension of radiolabeledgranulocytes, the statement regarding selectivegranulocyte labeling made by Peters et al. may not besupported. Kelbaek et al. (113) had noted that 11% ofthe ‘Tc-HMPAO activity is attachedto lymphocytesand monocytes, when both cell types constitute on]y6% of the differential counts.

Potential Pitfalls With Radiolabeled hukocytesThere has been concern that the sensitivity of “lln-labeled leukocytes may be affected by antibiotictherapy and chemotherapeutic agents. For antibiotics,these factors could reduce leukocyte chemotaxis bykilling or suppressing the bacteria and also directlyaffecting the leukocyte itself (114). In addition, invitro studies have indicated that some chemotherapeuticagents decrease WBC chemotaxis (115,116). Otherfactors that may potentially cause false-negative WBCscans by affecting leukocyte chemotaxis includesteroids, hyperalimentation, hemodialysis, andhypergl ycernia. However, clinical studies of lllln-labeled leukocytes in patients with the aforementionedfactors show no significant decrease in the sensitivityof the leukocyte scan (114,117-119).

In addition to potential problems involving false-negative results, lllIn-labeled leukocytes may presentoccasional false-positive uptake in tumor. In a study of249 patients, Fortner et al. (120) found that 12% ofpatients with known tumor at the time of imaging hadfalse-positive studies. The mechanism of leukocyteuptake in tumor is not completely understood. It maybe due to tumor necrosis, free iridium bound totransferring, labeling of lymphocytes in a mixed cellsample, or presence of a variable number ofinflammatory cells in certain tumor types. Othercauses of false-positive studies with Iabeld leukocytesinclude hematoma, early fracture, muscle contusion,and swallowed leukocytes.

However, the major concern, especially with theincreasing acquired immune-deficiency syndrome(AIDS) incidence in the past few years, has to be

either infection ofimmune-deficiencyduring the labeling

health care workers with humanvirus (HIV) from a needle stickprocess, or the possibility of the

misadministration of HIV-infected blood cells to a non-HIV patient. This latter concern has, in fact, recentlyoccurred.

For these reasons, there have been numerous effortsto develop a “kit preparation” so that the laboriousblood cell separation, labeling, and reinfection can beavoided. Other promising alternatives to radiolabeledautologous leukocytes have been used in clinical studieswith some success, such as ‘llIn-labeled nons~ificpolyclonal human immunoglobulin-G (IgG) and ‘Tc-labeled antileukocyte monoclinal antibodies.

RADIOLABELED ANTIBODIES

Recently there has been a great deal of interest inthe development of radiolabeled antibodies for thedetection of infection and inflammation.

Two approaches have been described for theradiolabeled antibody imaging of infections:

1. Antibodies directed against a surfacedeterminant of the infectious organism.

2. Antibodies directed against cells responding tothe infection such as white blood cells.

Although the concept for application of radiolabeledantibodies to infection/inflammation imaging appears tobe quite simple, there are some major difficulties whichmust be addressed prior to a wide clinical applicationof these non-neoplastic radioimmune imaging agents:

1. The antigen-antibody concentration at the lesionsite should be maximized.

2. The target-to-nontarget ratios in vivo should behigh.

3. The surface antigens should not be shed fromthe cell during the study.

4, Antibodies which cause lysis of cells, especiallyin the presence of complement, (i.e.,complement-mediated lysis), should not be used.

5. Human directed response against murineantibody (HAMA) must be nil.

lllIn-Labeled Nonspecific Polyclonal HumanImmunoglobulin-G

In 1988, Reburn, Fischman, and coworkers atMassachusetts General Hospital developed IgG for the

13

scintigraphic detection of foci of infection (121-125).Milligram quantities (0.025 mg per kilogram of bodyweight) of Sandoz IgG modified for intravenous usewere labeled with approximately 55.9 MBq (1.5 mCi)of ‘liIn via the cyclicanhydride diethylenetriamine-penta-acetic acid @TPA) method.

When compard with the currently availableinfection and inflammation imaging agents, ~~~In-IgGhas a number of advantages:

1. Early localization at the site of focalinflammation (the study can often be complettiwithin one day), which may lead to more rapiddiagnosis.

2. Unlike ‘7Gaand *Tc-HMPAO-]abeled WBCs,lllIn-IgG hw minimal bowel excretion;therefore, abdominal infection can be moreeasily identifid without the use of cathartics.

3. lilIn-IgG may detect nonbacterial infection(e.g,, fungus-caused infection), AIDS, andmalignancies, whereas radiolabeled leukocytesonly occasionally delineate these abnormalities.

4. The radiation dosimetry of ‘l’In-IgG permitsadministration of 55.5 MBq (1.5 mCi) liiInwith a radiation burden of < 40 mGy (4 rad)to the spleen.

5. IgG will be available as a kit, which is easierand quicker to prepare than labelingleukocytes.

6. Nordabeled IgG has already been approved forhuman use.

7. Since there is no need to withdraw a largevolume of blood for cell labeling, lilIn-IgGshould be quite suitable for pediatric patientuse.

8. No HAMA response is associatd with the useof ‘llIn-IgG since the IgG is obtained fromhumans.

However, l]lIn-IgG has four major limitations.First, ‘i*In-IgG has a higher background of normaluptake in the liver, spleen, and to a lesser extent, inthe urinary bladder. This may interfere with thediagnosis of infection in these areas. Second, thesensitivity of the l*lIn-IgG study is decreased inpatients with azotemia. Third, ll*In-IgG may localizein noninfected tumor sites, as does ‘7Ga-citrate,producing a false-positive result. Fourth, false-

negative results may occur in infections with littleassociated inflammation (l~~In-labeled WBCS may alsomiss these types of infections).

The exact mechanism of IgG uptake at sites ofinfection and inflammation is not fully understood.Many possible explanations have been proposed. First,nonspecific IgG binds to the efianced expression of theFc receptors in circulating macrophages, leukocytes, andlymphocytes in the pr=ence of focal inflammation. Thishypoth~is was based on animal experiments showinggood localization of intact IgG and Fc fragmenti, butminimal localization of Fab fragments at the site ofinflammation (12 1). However, studies by Oyen et al.(126) and Juweid et al. (125) provide suff~cient evidenceto reject this hypothesis. Oyen et al. (126) havedemonstrated fiat l’]In-IgA, an immunoglobulin tha~contains hardly any Fc receptors and which lacks Fcgamma receptor affinity, also accumulated in infectiousfoci. When IgG was treated with endoglycosidase-F toremove carbohydrates from IgG, in vitro Fe-receptorbinding was markedly decreased, but localization at sitesof infection was unaffected (125). Second, localkationof IgG in sites of infection may be through the bindingto bacterias (127). Although this may be a possibleexplanation for the mechanism of uptake, it does notexplain IgG uptake at sites of fungal infection or sterileinflammation. Third, it is like]y that increased capillarypermeability, which is a hallmark of inflammationcausing redness and swelling, probably plays the majorrole. However, studies comparing labeld *Tc-humanserum albumin and b7Gawith IgG showed greater IgGaccumulation as early as three hours post-injection (124).Therefore, this phenomenon may not be due solely toincreased blood flow and permeability at sites ofinflammation.

Although the mechanism for uptake of nonspecificIgG remains obscure, Fischman et al. (128) havesuggested that the Fc portion of IgG is the imagingfragment of choice in detecting hepatic and spleniclesions and in imaging vascular infection since the Fcfragment has approximately 50% lower accumulation inthe liver and spleen than IgG, and it also clears horn theblood pool more rapidly than IgG. They furtherspeculate that it is possible that other Fc fragments, suchas mFc, SFC, or stFc (128), may be more effectiveimaging agents. As of this time, labeled nonspecific IgGis not an FDA-approved agent.

-c-Labeled Monoclinal Antigranulocyte Antibodyme-Labeled BW 250/183)

A “neutrophil specific” rnonoclonal antibody wouldhave a greater potential for clinical application thannonspecific l’lIn-IgG because of higher specificity aswell as the sensitivity associated with monoclinalantibodies when compared to polyclonal antibodies such

m

o

●

●

14

as IgG, which cannot discriminate inflammation fromother diseases with increas~ capillary permeability.The drawbacks of the murine monoclinal antibodydirectd to a leukoc~e antigen are the lack of FDAapproval in the U.S. and the possible development ofHAMA response in patients.

An antigranulocyte IgG monoclinal antibody (MAbBW 250/183) was developed by Behringwerke(Marburg, Germany). This antibody is directti at anonspecific cross-reacting antigen (NCA-95), a 95 kDsurface glycoprotein, present on almost all humangranulocyte surfaces, which allows the antibody tointeract with “almost 95% of all granulocytes” (129).Despite the strong afinity between MAb BW 250/183and the granulocyte membrane, there is no occurenceof either antibodydependent or complement-dependentlysis of cells (130). Joseph et al. (131,132) labeled 75to 400 mg of monoclinal antibody with 296 MBq (8mCi) ‘Tc using the Schwarz method. In orderto achieve an efficient labeling of the antibody with‘Tc, the Schwarz method calls for the reduction ofantibody disulfide bonds to free thiol groups, to whichreduced ‘Tc binds. The reduced antibody waslyophil ized in the presence of the phosphatebuffer (pH 7.2). The labeling of the antibody with‘Tc is similar to a kit procedure for prepar-ing *Tc-labeled radiopharmaceuticals. First, thereduction of *TcO~ is achieved by using SnC12obtained from any commercial y available kits, such asPYP @yrophosphate), DTPA, or MDP (rndronate).The reduced ‘Tc then binds to the antibody. The LEhas been reported to be greater than 95% (133).

The half-life for disappearance of *Tc-labeled BW250/183 from the blood is approximately six hours.The initial uptake in the lungs clears by four hours.Approximate y 17% of the injected dose remains in theliver, and approximately 10% of the activity is seen inthe spleen at five hours after administration. Activityin bone marrow is always visible. At six hours post-injection, approximately 25% of the circulatingradioactivityy is bound to the granulocytes. Thisnumber is not high enough to be consistent with amonoclinal antibody that is supposed to be highlyspecific for the blood cells.

In their studies, Joseph et al. (131) have foundthat 10% to 20% of the patients showed a HAMAresponse with block potential, whereas about 20% to30% of the patients had a detectable HAMAresponse without blocking potential. Joseph et al.(131) indicate that the HAMA response is mostly ofthe IgM type, which is very weak and is detectabletwo to ten months after the scan.

Becker et al. (134) have observed that therecovery rate of ‘mTc-BW 250/183-labeled

granulocytes was less than 15% at 90 minuks, whichis much lower than that observed for lllln-oxine-labeled leukocytes and ‘Tc-HMPAO-labeledleukocytes. This may indicate that the localization ofthis antibody at foci of infection may not be primarilyattributable to binding to the NCA-95 surfaceglycoprotein in vivo. McAfee et al. (135) suggest thatthe free MAb BW 250/183 may migrate to aninfectious focus and then bind to viable or damagedgranulocytes that are already localized there.

OTHER AGENTS

*Tc-Labeled NanocolloidIn 1986, human albumin colloid particles with a

mean diameter of 30 nm (Nanocoll, Solco Basel,Switzerland) were labeled with technetium-99m(’%Tc-labeled nanocolloid) (Figure 8) and werereported to be effective in the detection of infectionand inflammation (136). A possible mechanism foruptake of ‘~Tc-nanocolloid in foci of infection are the

small colloidal ptiicles which freely diffuse throughthe increased leaky vascular endothelium andbasement membrane at the infectious/inflammatorysite.

Microaggregatedalbumin colloid

b 4

Sulfur colloidNanocolloid b

b +

0.01 0.1 1“”4

I?iiure 8. Size ranges for three different pafiulateradlopharmaceuticals whmh are used forimaging of kfectious and inflammatorylesions.

This localization process is similar to microcolloidsof serum albumin with a larger particle size (0.2-0.3~m). Due to the smaller size of *Tc-labelednanocolloid, the blood clearance is much faster than withmicrocolloids. Thus, imaging of ‘Tc-labelednanocolloid can be completed within four hours.Thereaf&er, activity may appear in the gastrointmtinaltract which may be attributed to spontaneous oxidation.

‘Tc-labeled nanocolloid has been thought to beuseful in the detection of osteomyelitis (137) and

15

suspected bone or joint disease (138,139), whereas thisagent has been shown to be less sensitive than lilInleukocytes for the assessment of IBD (140,141) andsoft tissue abscess or prosthetic vascular graft infection(138). *Tc-labeled nanocolloid may provide thequickest answer, but cmnot differentiate betweensterile and septic inflammatory lesions. This agent isnot approved for use in the USA by the FDA.

lllIn Labeled Avidin-BlotinRecently streptavidin or avidin, both of which are

xenoproteins, have been proposed by Rusckowski etal. (142-144) as alternatives to radiolabeled IgG forimaging infection. The nonspecific accumulation ofIgG has been attributed to increased vascularpermeability (145), and Rusckowski et al. (144) havereasoned that the protein streptavidin may exhibit thesame nonspecific mechanism of uptake as IgG. Theresults in their study (144) using a mouse modelwith an E. coli infection show that both 1llIn-labeledIgG and IllIn-labeled strepavidin have similar levelsof localization at the site of infection. Thisobservation strengthens the suggestion that theaccumulation of radiolabeled IgG may result fromnonspecific diffusion through leaky capillaries (145).

However, a common drawback in the use of bothlabeled IgG and streptavidin is the excess uptake inliver, spleen, and kidneys, and the slow clearance ofactivity from blood, both of which result in lowtarget-to-background ratios. In order to furtherimprove the target-to-background ratios,Rusckowski et al. (144) have used unlabeledstreptavidin to “pretarget” the abscess before theadministration of radiolabeled biotin, a relativelysmall molecule with high affinity (~ is approxi-mately 1015)(146) for both avidin md streptavidin.This high affinity of biotin for streptavidin and therapid blood clearance of unlabeled biotin improvedthe infected thigh-to-normal thigh ratio by threefold, and the infected thigh-to-liver and blood ratiosby eight fold in comparison with labeled IgG orlabeled streptavidin (144). If the pretargetingavidin-biotin concept for localizing infection can besuccessfully applied in humans, the lowerbackground levels of radiolabeled avidin-biotinwould allow an earlier imaging time. However,there is evidence that avidin and streptavidin areimmunogenic in humans, even at low doses (147).These agents are not yet FDA approved.

CONCLUSIONSA number of radiopharmaceuticals are available for

diagnosing infectious and inflammatory lesions. In the

past, the radionuclide of choice for the detection ofoccult infection has been b7Ga. However, b7Gais notspecific for abscess localization, as it also localizw intumor. In addition, ‘7Ga-citrate h~ other disadvantages(e.g., poor target-to-nontarget ratio, normal bowelactivity). Therefore, the radiolabeled leukocytetechnique has been developed and is increasingly appliedto infection/inflammation imaging. The use of lllln-labeled leukocytes has been well establish in the fieldof nuclear medicine and has allowed a number ofinfectious and inflammatory processes to be investigated.In fact, the use of ll*In-labeled WBCS has become thegold standard for imaging infectious and inflammatoryfoci. Recently, a number of newer *Tc-labeledradiopharmaceuticals (e.g., *Tc-~bumin colloid and‘Tc-HMPAO) have been used for leukocyte labeling.These ‘Tc-labeled WBCS require less time betweenreinfection and imaging and have demonstrated goodsensitivityy in localization of lesions. Another new areaof investigation is the use of radiolabeled antibodies(e.g., ‘*lIn-IgG and ‘Tc-labeled BW 250/183) for thedetection of infection and/or inflammation, which hasshown improvd sensitivity and specificity. Finally,‘Tc-labeled nanocolloids and IllIn-labeled avidin-biotinare also being studied as potential infection/inflammationimaging agents.

The currently available agents which have FDAapproval for the detection of infectious and/orinflammatory lesions are b7Ga citrate and Ii*In-labeledleukocytes. b7Gacitrate is best used when an FUO issuspected, as it localizm not only in areas of acute andchronic infection but also in tumors and chronicgranulomatous disease, all of which may cause an FUO.‘7Gahas also been shown to be superior to IllIn-labeledleukocytes in the evaluation of disc space infection.When single photon emission computed tomography isrequired, b7Ga is also the agent of choice due to thelarger amount of activity that can be administered, Thisis especially true when malignant external otitis issuspected. IllIn-labeled leukocytes are preferred wheninfection or inflammation is suspected in soft tissues orthe musculoskeletal system. For musculoskeletal sepsis,a combination of bone scintigraphy and l*]In-labeledleukocytes is recommended for non-marrow containingbone, and a combination of ‘i*In-labeled leukocytes anda marrow imaging study (usually with *Tc-sulfurcolloid) is recommended in marrow-containing regionsin order to help differentiate the normal leukocytedistribution within marrow from sites of infection orinflammation. Some investigators believe that a puregranulocyte preparation should be used when studyingsuspected vascular graft infection, as labeled plateletscan localize on foreign surfaces, although work withmixed cell populations has shown good sensitivity andspecificity. A purified granulocyte preparation has also

16

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*

●

o

been recommended for the evaluation of inflammatorybowel disease, although again both the ‘liIn and *Tcmixed cell preparations have shown good resultsclinically.

The use of *Tc-HMPAO-labeled leukocytes hasbeen increasing rapidly. The use of ~TC-HMPAO tolabel leukocytes is not yet FDA approved. This agentwill be mpecially usefil in children, where radiationdosimetry is a concern with the “’In-labeledleukocytes. With the ‘mTc-HMPAO-labeld WBCS,special concern is needed in evaluating sites ofabdominal infection and inflammation, as the normaldistribution of *Tc-HMPAO-labeled leukocytesincludes a significant component of bowel activity aftertwo to four hours. Once the newer indication for*Tc HMPAO is approved, this agent may havesignificant benefit over our currentradiopharmaceuticals. Some articles (148, 149) haveaddressed the issue as to the use of approved drugs ina nonapproved way (e.g., unlisted indication, dosage,route of administration, or age group); it is generallybelieved that the unapproved use of approved drugsshould be considered as part of the practice ofmedicine provided the physician is well informedregarding the drug and that the unapproved usage bebased on firm scientific rationale.

Finally, it is important to remember that in bothinfectious and non-infectious inflammatory lesions thesame pathophysiology is present. Therefore, it isunlikely, ~at- witl---utilization of either currentlyapproved agents or agents now under investigation forfuture use that one will be able to differentiate with anydegree of accuracy infectious from non-infectiousinflammatory proce;ses.

ACKNOWLEDGEMENTS

The authors would like to thank Doctors Mary F.Hauser and Paul A. Thomas, Nuclear Medicine,Department of Diagnostic Radiology, Mayo Clinic, fortheir useful comments on this paper. We also thankMs. Vicki S. Krage, Ms. Rose M. Busta, and Ms.Carole M. Geizura for their skillful assistance in themanuscript preparation.

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QUESTIONS

1. The oxidation state of ‘7Ga in the chemicalstmcture of b7Ga-citrate is:

A. 1+B. 2+c. 3+D. 4+

2. Which of the following modalities offers thefastest and least expensive mode for thedetection of abscesses?

A. computed tomographyB. magnetic resonance imagingc. ultrasoundD. radionuclde scintigraphy

23

3. What is the optimal pH of the labelingmedium for radiolabeled leukocytes?

A. 4.5B. 6.5c. 8.5D. 10.5

4. Why is hetastarch added to the whole bloodin the process of radiolabeling leukocytes?

A. to improve the labeling efficiencyB. to reduce microaggregation of

leukocytes during labelingc. to remove smaller and lighter

plateletsD. to increase the RBC sedimentation

rate

5. Which of the following iridium chelatingagents has been approved by the FDA?

A. ACDB. MERCc. oxineD. tropolone

6. What is the mean particle size for ‘gmTc-Iabeled nanocolloids?

A, 3nmB. 30 nmc. 300 nmD. 3000 nm

7. The pretarget approach used in the l**ln-labeled avidin-biotin is:

A. to improve the target-to-nontargetratios

B. to increase the labeling efficiencyc. to reduce HAMA responsesD. to allow higher administration dose

8. Which of the following quality controlmethods is the in vivo test for radiolabeledleukocytes?

A. chemotaxisB. Ficoll-Hypaque distributionc. CBCD. cell recovery

9. The standard dose of l*lIn-labeled lgG for thescintigraphic detection of infection/inflamma-tion foci is:

A. 0.05 mCiB. 0.5 mCic. 1.5 mCiD. 2.5 mCi

10, Which of the following structural formulasrepresents 1~lIn-oxine?

A. lllln (Oxine)B. 1*‘In (Oxine)zc. 11‘In (Oxine)~D, lllIn (Oxine)q

11. Which of the following is the reason for usingPercoll in the radiolabeling process?

A. to separate pure granulocytesB. to remove LRPRPc. to reduce plasma in the final

preparationD. to promote aggregation during labeling

12. When radiolabeling leukocytes in vitro, themost suitable incubation temperature is: o

A. 4°CB. 10”Cc. 25°CD. 41°c

13. In order to have an efficient radiolabeling ofMAb BW 250/183 with ‘~c, the Schwarzmethod calls for which of the following 9@Tcspecies to bind with the antibody?

A. reduced ‘WCB. hydrolyzed reduced ‘Tcc. ‘gmTc-PYPD. ‘hTc@

14. Which of the following radiopharmaceuticalsmay develop a HAMA response in patients?

A. lllIn-labeled IgGB. 11lIn-labeled avidin-biotinc. 99mTc-labeled nanocolloidsD. ‘Tc-labeled BW 250/183

24

—

,

15.

<

0

16.

17.

●18.

19.

20.

0

In WBCS radiolabeled with ‘hTc-HMPAO,this agent is able to diffuse through cellmembrane due to itscharacter.

A. symmetricB. lipophilicc. hydrophilicD. ionic

Polysaccharide helps to clump RBCS togetherand reduce the required sedimentation timeby:

A. 1/5B. 1/4c. 1/3D. 1/2

Which statement best describes the outcomeof the final product if leukocytes are labeledwith l]lIn-oxine in the presence of plasma?

A. increased urinary excretionB. improved sedimentation ratec. decreased labeling efficiencyD. diminished chemotactic properties

The blood disappearance tl,z for ]l]In oxine-Iabeled leukocytes in humans is:

A. 2- 3,5 hrB. 5- 7.5 hrc. 10 -14,5 hrD. 22 -25.5 hr

Which of the following is the majorcomposition of ‘9Tc-HMPA0 in thespontaneous elution from labeled WBCS?

A. primary ‘9WC-HMPA0 complexB. secondary ‘g”Tc-HMPAO complexc. hydrolyzed-reduced ‘gmTcD. ‘hTc-pertechnetate

Both streptavidin and avid in are:

A. proteinsB. lipidsc. steroids

21.

22.

23.

24.

25.

Approximately times morecompleting agent than *llIn is required beforecell labeling will occur.

A. 5B. 10c. 50D. 100

The chemical name for oxine is:

A. 8-hydroxyquinolineB. 8-deoxyquinolinec. 8-methoxyquinolineD. 8-xenoxyquinoline

Which of the following radiopharmaceuticalshas a shelf life of only 30 minutes?

A, ‘gmTc-nanocolloidB. *llIn-tropolonec. ‘9”Tc-HMPA0D. l“In-IgG

Hydroxyethyl starch is commercially availablein % solution?

A. 2%B. 4%c. 6%D. 8%

Which of the following is not considered anadvantage when using lllIn-IgG for thelocalization of infection and/or inflammation?

A. 1l’In-IgG can detect nonbacterialinfection

B. lllIn-]gG may localize in tUmOr SiteS

c. 111In-IgG has minimal bowel excretionD. IgG is available as a kit

D. carbohydrates

0