Vol. 30, No. 8JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2071-20760095-1137/92/082071-06$02.00/0Copyright © 1992, American Society for Microbiology

Rapid Whole-Blood Microassay Using Flow Cytometry forMeasuring Neutrophil Phagocytosis

CATHY WHITE OWEN,12* J. WESLEY ALEXANDER,1'2 R. MICHAEL SRAMKOSKI,2 ANDGEORGE F. BABCOCK1' 2

Department of Surgery, University of Cincinnati College ofMedicine, 231 Bethesda Avenue, Cincinnat4Ohio 45267, 1* and Cincinnati Unit, Shriners Burns Institute, Cincinnati Ohio 452292

Received 18 February 1992/Accepted 19 May 1992

A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by humanneutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 I of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commerciallyavailable whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external freeor adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalizedbacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size,(ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability todistinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes asquenching agents, and (vi) ability to fix cells and store for future FCM analysis.

The clinical importance of neutrophil (polymorphonuclearleukocyte [PMN]) function, particularly with regard tophagocytosis of microorganisms, has been well established(1, 8, 10). The use of flow cytometry (FCM) with fluores-cently labeled particles (bacteria, yeast cells, or latex beads)has facilitated the investigation of this important PMNfunction (3, 7). Early FCM methods provided no reliabledistinction between internalized particles and those whichmay be simply adherent to the cell surface (3, 12). Morerecently, quenching of external fluorescence has been ac-complished with the use of vital dyes such as ethidiumbromide (EtBr) (9), trypan blue (2), or crystal violet (8, 13).The use of these dyes, however, is quite cumbersome,creates technical problems in FCM methods, and precludesthe use of fixatives which would otherwise allow delayedFCM analysis.

It is well known that pH changes may significantly affectthe fluorescence of fluorescein compounds, most notablyfluorescein isothiocyanate (FITC), which is a common agentused to label particles for phagocytic assays (4, 5, 6). In thisarticle, we describe a new FCM method which utilizesFITC-labeled Staphylococcus aureus bacteria and a 100-plwhole-blood sample requiring no leukocyte (WBC) separa-tion. After incubation of blood with bacteria, phagocytosis ishalted and erythrocytes (RBCs) are lysed with a formic-acid-based commercially available reagent (Immuno-Lyse; Coul-ter Corp., Hialeah, Fla.) which concurrently quenches thefluorescence of external nonphagocytized bacteria. The useof vital dyes is avoided, delayed analysis is possible, andonly small blood volumes are required.

MATERIALS AND METHODSLabeling of bacteria. S. aureus organisms were cultured

for 16 h at 37°C in Trypticase soy broth (Baltimore BiologicalLabs, Baltimore, Md.) with FITC (Research Organics, Inc.,Cleveland, Ohio) at a concentration of 30 to 50 jig/ml.Bacteria were then washed twice in Dulbecco's phosphate-buffered saline (DPBS) (GIBCO Laboratories, Grand Island,

* Corresponding author.

N.Y.) and heat inactivated at 60°C for 30 min. They wereagain washed and resuspended in DPBS to a final concen-tration of 109 bacteria per ml. The organisms were examinedby fluorescence microscopy for uniformity of FITC staining.Confirmation was provided by FCM. The bacterial suspen-sion was then aliquoted and frozen at -70°C in a light-protected environment.

Blood samples. Blood was obtained from 20 normalhealthy adult volunteers, collected in sodium EDTA, andstored at room temperature for no longer than 6 h prior touse. More prolonged storage resulted in suboptimal PMNisolation and altered phagocytosis (data not shown). TotalWBC counts were performed on a Neubauer hemacytometerby using 3% acetic acid lysis of RBCs. If the WBC countexceeded 107 cells per ml, the whole blood was diluted withDPBS to bring the WBC count within the range of 3.0 x 106to 9.9 x 106 cells per ml. When isolated PMNs were used forthe assay, as a comparison control for the whole-bloodassay, they were isolated in the following manner. Fivemilliliters of whole blood, collected in sodium EDTA, waslayered over a Ficoll gradient consisting of 4.4 ml of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and 0.6 ml of 85%Hypaque (57% meglamine ditrizoate-28% sodium ditrizoatein H20). The gradient was centrifuged at 500 x g for 30 minat room temperature. This process resulted in two clearlydefined bands of WBCs, the upper band consisting of mono-nuclear cells (including monocytes and lymphocytes) and thelower band consisting of PMNs and some minor RBCcontamination. The majority of the RBCs went through thegradient and pelleted at the bottom. Contaminating RBCswere removed by H20 lysis. This separation techniqueyielded a >98% pure population of granulocytes with >98%viability by trypan blue exclusion. The average PMN yieldwas approximately 65%. PMNs were collected and washedtwice in DPBS and resuspended to a concentration of 5 x 106cells per ml.

Phagocytosis assay. In 20 separate experiments, 100 ,ul ofwhole blood, diluted whole blood, or isolated PMNs wasdispensed into polystyrene tubes for each time point,namely, at time zero and at 2, 5, 10, and 15 min. Whole bloodor diluted blood samples were washed twice in DPBS to

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remove EDTA and plasma. Opsonization was accomplishedby resuspending cells in 100 ,ul of pooled normal human typeAB serum in each tube. After a 3- to 5-s sonication, 10 ,ul ofstock FITC-labeled bacterial solution (109 bacteria per ml)was added to each tube (10:1, bacteria/cells). Cytochalasin D(Sigma Chemical Co., St. Louis, Mo.) (final concentration, 5,uM) was added to the time zero tube immediately. Theremainder of the tubes were incubated for the appropriatetime periods in an agitating 37°C water bath. Phagocytosiswas stopped in each tube by the addition of cytochalasin Dat a final concentration of 5 pM. Tubes were placed imme-diately on ice, and 3 ml of ice-cold DPBS was added to each.For each blood sample, one tube containing cells only (nobacteria) was carried through the procedure as a control toevaluate the background fluorescence of the PMNs alone.Whole-blood lysis. All tubes were washed twice in cold

DPBS. RBCs were lysed with Immuno-Lyse (Coulter) byfollowing the prescribed protocol. Briefly, the lysis reagentwas diluted 1:25 with DPBS. One milliliter of this solutionwas then added to each tube. All tubes were briefly vortexedimmediately and at 1 min after addition of the reagent. Tubeswere maintained on ice, and at 2 min, the lysis was halted bythe addition of 250 ,ul of Coulter fixing reagent. Aftersufficient washing with DPBS to remove all free hemoglobin,the cells in each tube were either fixed with 0.5 ml of ice-cold1% paraformaldehyde solution containing 1% sodium ca-codylate and 0.8% NaCl or prepared for immediate FCManalysis by the addition of 0.5 ml of ice-cold DPBS. Para-formaldehyde-fixed samples were stored in a light-protectedenvironment at 4°C for future FCM analysis.Those experiments involving separated PMNs also in-

cluded treatment with Immuno-Lyse so that the effects ofthe process on adherent external bacteria and on isolatedPMNs could be evaluated. Again, cells were either fixedwith paraformaldehyde and stored or placed in DPBS forimmediate FCM analysis.FCM. FCM was performed with a Coulter Epics 753 FCM

(Coulter Cytometry) by using a 488-nm line of an argon ionlaser. Green fluorescence was collected by using a 530 + 15nm band pass filter and linear amplification. Data werecollected and analyzed by using the Coulter Easy-2 soft-ware. Non-PMN debris were excluded from analysis byusing forward and 90°-angle light scattering.Adherence versus phagocytosis. The effect of the whole-

blood lysis technique on external free bacteria was tested byprocessing free FITC-labeled bacteria alone with the Immu-no-Lyse reagents as described above. The effect of Immuno-Lyse on external membrane-bound bacteria was evaluatedby arresting phagocytosis with cytochalasin D before incu-bation of cells with the fluorescent S. aureus organisms.Briefly, whole blood, diluted whole blood, or isolated PMNswere pretreated with cytochalasin D (final concentration, 5,M) to inhibit phagocytosis. Bacteria were then added toeach tube, and the assay was carried out according to theprotocol. In this system, phagocytosis, not bacterial adher-ence, is inhibited. FITC fluorescence of PMNs was evalu-ated in tubes which did not undergo lysis (purified PMNsonly) and in tubes which were lysed (purified PMNs andwhole blood).These results were compared with the results obtained by

using the technique of Fattorossi et al. (9), in which EtBr isused to quench FITC fluorescence of free or membrane-bound microorganisms. Briefly, adherent or free FITC-labeled organisms were stained with EtBr (final concentra-tion, 50 ,ug/ml) for 5 min at 4°C after the phagocytosis assayhad been performed. By the process of resonance energy

CO)

0

Fluorescence IntensityFIG. 1. (A) FITC fluorescence intensity (FI) of heat-killed, son-

icated bacteria grown in Trypticase soy broth in the presence of 30pg of FITC per ml; (B) Fl of unsonicated FITC-labeled bacteria; (C)Fl of FITC-labeled bacteria after treatment with Immuno-Lyse.

transfer, FITC fluorescence was quenched. Since it is wellknown that the EtBr does not penetrate intact cell mem-branes, internal ingested bacteria cannot be affected byEtBr, and, thus, the bacteria retain their green fluorescence.Therefore, green fluorescence of any PMN after EtBrquenching can only be explained by the presence of internalingested bacteria.

RESULTS

FITC labeling of S. aureus. In the initial experiment, S.aureus was grown in broth containing 50 ,g of FITC per ml.The bacteria were brightly and homogeneously stained.They retained their fluorescence through the heat killingprocess and through several freeze-thaw episodes as well(data not shown). In later experiments, similarly good resultswere obtained when the bacteria were grown in as little as 30,ug of FITC per ml of broth (Fig. 1A). Cultures of S. aureus

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were labeled with FITC on six separate occasions. In allexperiments, the bacteria were adequately stained. How-ever, the intensity of staining did vary among cultures.Therefore, it is recommended that with each new culture,FITC fluorescence should be checked on the FCM and theinstrument should be recalibrated accordingly. Fluorescencewas stable for at least 3 months when suspensions werestored at -70°C in a light-protected environment. After thebacterial suspension had been thawed, a brief (3- to 5-s)period of ultrasonification was required before the introduc-tion of the bacteria into the phagocytosis system to break upclumps of bacteria which, on FCM, may be perceived aspoor or heterogeneous staining of bacteria (Fig. 1B). Thismethod of fluorescently labeling bacteria is also quite satis-factory for the gram-negative bacteria Escherichia coli (datanot shown).

Effect of Immuno-Lyse on free bacteria. When free FITC-labeled bacteria were subjected to the lysis process, it wasdiscovered that nearly all FITC fluorescence was quenched.Figure 1C demonstrates FITC fluorescence of these bacteriaafter treatment with Immuno-Lyse in one experiment. Therewas an average reduction of FITC fluorescence of 78 ± 3%(standard error of the mean) in all experiments after lysis. Byusing the EtBr quenching technique, there was an averagereduction of FITC fluorescence of 69 ± 6%. Thus, ourmethod compares favorably with that of Fattorossi et al. (9).These results were reproducible in 10 separate experiments.

Effect of Immuno-Lyse on isolated PMNs. Figure 2 illus-trates the effect of Immuno-Lyse treatment on Ficoll gradi-ent-isolated PMNs. This figure represents the results of oneexperiment which typifies the outcome obtained in 10 exper-iments. There were significant changes in cell size andgranularity as assessed by light scatter histograms afterImmuno-Lyse treatment of these cells (Fig. 2B) when com-pared with isolated PMNs which had not undergone lysis(Fig. 2A). A similar effect was not seen after whole-bloodlysis (Fig. 2C). PMNs from whole-blood lysis fell intoessentially the same gate as purified PMNs which have notundergone lysis treatment. This would suggest that the useof Immuno-Lyse on purified PMN populations is not advis-able and that, indeed, a whole-blood assay is preferable.However, to further clarify this effect of Immuno-Lyse onisolated PMNs, a series of experiments was performed inwhich various volumes of lysis reagents were used. Figure 3shows the results of these experiments. Figure 3A againdemonstrates the dramatic effect of Immuno-Lyse on iso-lated PMNs when 1.0 ml of lysis reagent was used. Figures3B and C show the effects of using 0.5 ml or 0.25 ml of lysingreagent, respectively. Clearly, the effect on cell size andgranularity is almost completely obliterated with the use ofless lysis reagent. The use of these lower volumes of lysisreagent, even as little as 0.25 ml (data not shown), had noeffect on the ability to quench external bacterial FITCfluorescence. Therefore, if Ficoll gradient-isolated PMNsmust be used, reduction of the volume of Immuno-Lyse isstrongly advised.

Effect of Immuno-Lyse on membrane-bound bacteria. Ta-ble 1 indicates the percentage of PMNs which have cell-associated FITC fluorescence at each time point (time zeroand at 2, 5, and 10 min) in 10 phagocytosis experimentswhich involved either purified PMNs which were not treatedwith Immuno-Lyse (group A), purified PMNs which weretreated with Immuno-Lyse (group B), and purified PMNswhich were prevented from phagocytizing with cytochalasinD and were not treated with Immuno-Lyse (group C).Cell-associated FITC fluorescence in the group A experi-

C')

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FALSFIG. 2. (A) Light scatter histogram of purified PMNs before

treatment with Immuno-Lyse. (Note the small agranular populationof cells represents contaminating RBCs.) (B) Histogram of purifiedPMNs after Immuno-Lyse treatment. (Note the significant change incell size [FALS] and granularity [9OLS].) (C) Histogram of wholeblood after Immuno-Lyse treatment. (Note a slight change in cellsize but granularity is preserved.) This sample also contains lym-phocytes and monocytes.

ments was the result of both phagocytized and adherentbacteria, since these cells did not undergo treatment withImmuno-Lyse. Fluorescence in the group B experimentswas the result of internalized bacteria only, while fluores-cence in group C, the arrested phagocytosis group, resultedfrom membrane-bound bacteria only.

Theoretically, the sum of the results of groups B and Cshould approximate the percentage of fluorescent cells ingroup A for each time point. Again, Table 1 demonstratesthis comparison. Indeed, the value for groups B+C appearsto approximate that of group A fairly well. However, valuesfor B+C do tend to be slightly lower than A values at eachtime point. Because of the previously mentioned effect of

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00

FALSFIG. 3. Effect of various volumes of Immuno-Lyse on the light

scatter histogram of Ficoll gradient-isolated PMNs. (A) A 1.0-mlvolume of Immuno-Lyse was used. (Note the dramatic change incell size [FALS] and granularity [90LS].) (B) An 0.5-ml volume ofImmuno-Lyse was used. (C) An 0.25-ml volume of Immuno-Lysewas used. (Note that granularity and cell size are preserved.)

Immuno-Lyse on purified PMNs (see above), we hypothe-size that both B and C values may be artificially loweredsince some of the fluorescent cells may not fall within thePMN gates on FCM after lysis. To reiterate this point, Table2 compares the percentages of FITC fluorescent cells ob-served at each time point for purified PMNs which weretreated with Immuno-Lyse with those ofwhole blood treatedwith Immuno-Lyse. These data support the hypothesis thatImmuno-Lyse has the same effect on membrane-boundFITC-labeled bacteria as it does on free fluorescent organ-isms. Namely, Immuno-Lyse treatment extinguishes FITCfluorescence of external adherent bacteria.When the Immuno-Lyse method was compared with the

TABLE 1. Percentage of PMNs with FITC fluorescenceafter phagocytosis

% PMNs with FITC fluorescence (±SEM)aTime

Group A Group B Group C Group B+C

Time zero 13 2 6 1 4 2 10 22minT2 17 4 6 2 4 1 10 45 min T5 28 3 8 4 19 3 27 310minT10 63±2 25±3 32±5 57±6

a Group A, purified PMNs which did not undergo lysis assay; group B,purified PMNs which did undergo lysis assay; group C, purified PMNs inarrested phagocytosis assay which were not treated with RBC lysis reagent.Groups B+C should approximate the percentage of FITC fluorescent cells ingroup A for each time point. SEM, standard error of the mean.

method of Fattorossi et al. (9), which uses the vital dye EtBrto quench FITC fluorescence of external membrane-boundbacteria, favorable results were again obtained (Table 3). InTable 3, the percentages of cells demonstrating cell-associ-ated fluorescence in three groups are compared: group I,purified PMNs which were not treated with Immuno-Lyseafter phagocytosis; group II, treated in the same manner asgroup I but quenched with EtBr prior to FCM analysis; andgroup III, PMNs from the whole-blood Immuno-Lyse assay.A similar, if not somewhat greater, degree of FITC quench-ing is seen with the Immuno-Lyse method, thus confirmingthat this method does allow differentiation of intracellularphagocytized organisms from external membrane-boundbacteria.

Ability of assay to detect phagocytosis. Figure 4 typifies thehistograms obtained from 10 experiments using normalPMNs in this assay. Figure 4A demonstrates the fluores-cence intensity (FI) at time zero, while Fig. 4B, C, and Ddemonstrate the FI at 2, 5, and 10 min, respectively. By 10

min, maximal FI is obtained when normal PMNs are used.However, PMNs from a variety of clinical patient popula-tions may vary in terms of the time when maximal FI isobtained (data not shown).

Effect of paraformaldehyde fixation on use of quenchingvital dyes. When EtBr was used to quench bacterial FITCfluorescence in unfixed PMNs, our results were consistentwith those of Fattorossi et al. (9), i.e., there was adequatequenching observed. However, when previously paraform-aldehyde-fixed cells were treated with EtBr, no suchquenching occurred. In fact, there was a dramatic increase inwhat was read as FITC fluorescence in cells treated in thisway. These results would indicate that the use of quenchingvital dyes such as EtBr after fixation with paraformaldehydeis not possible, contrary to the findings of Fattorossi et al.

Reliability of results after paraformaldehyde fixation in thewhole-blood Immuno-Lyse method. Comparisons of results

TABLE 2. Effect of Immuno-Lyse on purified PMNs versuswhole blood

% Fluorescent PMNs (±SEM)rTime Immuno-Lyse-treated Immuno-Lyse-treated

purified PMNs whole blood

Time zero 6 ± 2 3 ± 12min 6 ± 1 4 ± 25 min 8 ± 3 17 ± 310 min 25 ± 3 46 ± 6

a SEM, standard error of the mean.

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obtained immediately after the assay in unfixed cells andassays which were fixed and delayed (up to 3 months)demonstrated virtually no variation in results. All delayedresults fell well within ±2 standard deviations of initial meanvalues of percentage of cells with FITC fluorescence.

DISCUSSION

Traditionally, FITC labeling of bacteria has been accom-plished by the addition of FITC to heat-killed organisms,followed by a relatively prolonged incubation to ensure evenand adequate staining. We have been able to eliminate theneed for this step by adding the FITC directly to the mediumat the time of inoculation with bacteria. Good results wereobtained when the bacteria were grown in the presence of aslittle as 30 ,ug of FITC per ml of medium. Fluorescence wasstable for at least 3 months when suspensions were stored at-70°C in a light-protected environment. The use of Immuno-Lyse in a whole-blood phagocytosis assay solved many ofthe problems associated with other FCM phagocytosis meth-ods. Most methods require the isolation of PMNs by usingdensity gradient centrifugation, which has been shown byseveral authors to alter morphologic as well as functional

TABLE 3. Percentage of FITC fluorescence cells afterphagocytosis in purified PMNs not treated with Immuno-Lyse

(group I), EtBr-quenched purified PMNs not treated withImmuno-Lyse (group II), and PMNs from whole-blood lysis assay

(group III)

% FlTC fluorescence cells (±SEM)a in:Time

Group I Group II Group III

Time zero 13 ± 3 14 ± 3 3 ± 12 min 17 ± 1 12 ± 1 4 ± 25min 28 ± 5 15 ± 1 17 ± 310 min 63 ± 2 52 ± 7 46 ± 3

a SEM, standard error of the mean.

properties of PMNs and which allows for potential uncon-trolled selection of subpopulations of PMNs (15-17). It is,therefore, desirable to avoid these separation techniqueswhenever possible. The method described in the presentarticle excludes the need for PMN separation by Ficolldensity gradients or dextran sedimentation and allows theuse of very small volumes of sample blood, thus providing afeasible way to study small volumes of blood such as thoseobtained from pediatric populations.

Additionally, the use of Immuno-Lyse allows for thediscrimination between external and internal bacteria, thusproviding an accurate assessment of true phagocytosis. Theuse of vital dyes as quenching agents requires an additionalincubation period and may, in some circumstances, foul thetubing and flow cell of the instrument. This can result inunwanted quenching of subsequent sample runs, and exten-sive cleaning of the tubing and flow cell is required after suchexperiments.

Cantinieaux et al. (8) have described the use of paraform-aldehyde as a cell fixative to allow delayed FCM analysis, atechnique which is quite useful in a busy laboratory or wherea core FCM facility is utilized. However, our attempts to useEtBr to quench FITC fluorescence in cells fixed with para-formaldehyde were totally unsuccessful. The fact that para-formaldehyde fixation permeabilizes the PMN cell mem-brane, as evidenced by extensive intracellular staining withEtBr, prevents the use of this agent for fixation. Because anintact cell membrane is essential to the principle of EtBrquenching of external fluorescent bacteria, it cannot be usedas a quenching agent in paraformaldehyde-fixed cells. In-deed, there was an increase in fluorescence when EtBr wasadded to paraformaldehyde-fixed cells. This was most likelydue to excessive intracellular EtBr staining. Although thelatter fact would not be a problem with other vital dyes suchas crystal violet or trypan blue, the effect of paraformalde-hyde on the integrity of the PMN cell membrane wouldobviate the use of these agents when paraformaldehydefixation is planned. These problems are completely elimi-

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nated by using this methodology. Paraformaldehyde fixationand delayed FCM are advantages of this methodology.

Preliminary experiments have indicated an additional ad-vantage of this technique. Since whole-blood lysis FCMtechniques have been used for several years to identify cellswhich react with these fluorescently labeled monoclonalantibodies, cell surface staining with monoclonal antibodieslabeled with fluorochromes other than FITC can be used inmulticolor fluorescence procedures. By using a double- ortriple-color immunofluorescence FCM method with phyco-erythrin, Texas red, or allophycocyanin, we have been ableto assess the phagocytosis of PMNs which have previouslybeen stained with fluorescent monoclonal antibodies, thusevaluating the phagocytosis of subpopulations of PMNswithout time-consuming panning or cell-sorting methods.One disadvantage of this technique, as with other methods

which have used bacteria, is that the actual number ofmicroorganisms ingested by each cell is difficult to assess. Itshould be noted that as bacteria are phagocytized, oxidativemetabolic activity is initiated and phagolysosome acidifica-tion begins to occur. This will result in a decreased intensityof FITC fluorescence emitted by those bacteria containedwithin the phagolysosomes. In normal adults, it has beenfound that this effect becomes evident sometime betweentime zero and 15 min in this assay. A moderate degree ofindividual variability exists and may be more evident inill-patient populations. Thus, it is advisable to use severaltime points when performing this assay to determine thepoint at which maximal intracellular fluorescence occurs.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the Shriners ofNorth America.We thank Lois Marchionne for her assistance in preparing this

manuscript and Chris Haviland for technical assistance.

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