J O U R N A L OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 5 243-250 (1990)

A Chemiluminescence Assay for E r y t hrophagocytosis I. D o w n i n g * , J. G.Templeton, R. M i t c h e l l a n d R. H. Fraser Glasgow and West of Scotland Blood Transfusion Service, Law Hospital, Carluke, Lanarkshire, Scotland ML8 5ES. UK.

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized w i t h anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 PI,light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35-45 minutes, the reaction being exhausted by 120 minutes. Determination o f the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5 % and 13 % respectively. We found the chemiluminescent response t o be affected by the number of erythrocytes used in the assay and by the composition of the medium i n which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay t o a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier t o perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error. Keywords: Mononuclear leukocytes; monocytes; cherniluminescence; anti-D IgG;

erythrophagocytosis INTR 0 DUCTlO N Erythrocytes coated with non-complement binding IgG have been shown to bind to peripheral blood mononuclear phagocytes via the Fcy receptor, (Von Dem Borne et al., 1977; Logue and Rosse, 1976; Van Der Meulen et al., 1978). This gives rise to rosette formation and subsequent phagocytosis or extracellular lysis of the erythrocyte (Engelfriet et al., 1981). In patients with autoantibodies (e.g. autoimmune haemolytic anaemia) this can cause a pathologic, problem. The provision of blood to these patients and others who are direct antibody

positive or who have alloantibodies against high incidence antigens associated with variable erythrocyte destruction can be difficult owing to the sometimes uncertain clinical status of these antibodies. There are several possible methods of determining erythrocyte survival in such cases, with much recent work focusing on the ability of mononuclear phagocytes to phagocytose or rosette appropriately sensitized erythrocytes (Schanfield et al., 1981; Douglas et al., 1985; Nance et al., 1987). However, this technique is not without problems and requires an experienced observer if the microscopy is to avoid subjective errors. An alternative

* Author for correspondence OSS4-3996/90/040243-08 $05.00 0 1990 by John Wiley & Sons, Ltd

Received January 1990

244

I. DOWNING, J. G. TEMPLETON,

approach now being examined is to use the associated events linked with the occupation of an Fcy receptor. The predominant effect of these events is the release of large amounts of reactive oxygen intermediates (Weiss et al., 1978; Weiss and La Buglio, 1982; Geffner et al., 1987) which in the prescence of luminol can be converted to light (Easmon et al., 1980), providing the basis of an assay for phagocytic activity. We have developed such a chemiluminescence assay based on this approach and have compared it with the microscopic assay in its ability to detect erythrocytes sensitized with an anti-D immunoglobulin preparation.

METHODS Isolation and Storage of Leukocytes

Mononuclear leukocytes were prepared from a leukocyte fraction obtained from healthy blood donors by apheresis using a Haemonetics V-50 plasmapheresis unit adjusted for lymphocyte collection. Mononuclear cells were purified and frozen using a modification of the methods of B ~ y u m (1983) and Weiner (1976). The leukocyte fraction was initially centrifuged at 500 g for 10 minutes to remove excess platelets. The cells were then resuspended to their initial volume in RPMI 1640 cell culture medium (Imperial Laboratories, Staffordshire, UK) and 6 ml aliquots layered over 5 ml Lymphoprep (Nycomed UK Ltd, Birmingham, UK). After centrifugation at 500 g for 20 minutes, the leukocyte layer was removed by Pasteur pipette, mixed with its own volume of RPMI 1640 supplemented with 10% heat inactivated foetal calf serum (FCS) (Sigma, Poole, UK) and centrifuged again for 5 minutes at 500 g. The cells were resuspended in cold RPMI 1640 Searle modification without phenol red (RPMI-SM) (Northumbria Biologicals Ltd, Northumberland, UK) to give a count of approximately 80 x lo6 cells per millilitre. Following the addition of FCS to 50% (v/v) an equal volume of cold RPMI-SM 20% DMSO (BDH Chemicals Ltd, Poole, UK) was added slowly on ice with gentle stirring. The cells were then dispensed into vials in 1 ml aliquots containing approximately 20 x lo6 cells and cooled to - 80 "C in a polystyrene container before storing in liquid nitrogen.

R. MITCHELL AND R. H. FRASER

Assessment o f monocyte preparation

The number of monocytes present in a mononucleaf leukocyte preparation was assessed using the method of Hogg et al. (1984) utilizing a mouse monoclonal anti-monocyte antibody UCHM-1 (SAPU, Carluke, UK) as the first antibody and a sheep FITC conjugated anti-mouse gamma globulin (SAPU, Carluke, UK) as the second antibody. The results were quantitated by fluorescence microscopy using an Olympus BH-2 microscope system. In addition a sample of cells from each batch was prepared on a slide and stained with the Diff-Quik stain system (Travenol Laboratories Ltd, Newbury, UK), a modified Giemsa-May-Grunwald stain and visually checked for the presence of granulocytes, particularly neutrophils. However, none have been detected after freeze-thawing. Viability was determined by phase contrast microscopy and acridine orange-ethidium bromide fluorescence (Absolom, 1986).

Sensitization of erythrocytes

Erythrocytes (group 0) were obtained from the random voluntary donor panel (Glasgow & West of Scotland Blood Transfusion Service). A 1 % suspension of cells (phenotype DCcEe, Fisher notation) were incubated with human anti-Rh(D) immunoglobulin (Protein Fractionation Centre, Edinburgh, UK, Lot 100470120, 262 IU/ml) 1/50 dilution, 5.25 IU (except for titration experiments when dilution varied) for 60 minutes at 37 "C in low ionic strength saline. Unsensitized cells, as controls, were included with each batch of sensitized cells. In addition ddccee phenotype erythrocytes were pretreated with the anti-Rh(D) immunoglobulin. Cells were washed three times, made up to 1 ml and an aliquot counted on a Coulter S560 Haematology Analyser before suspending at 12 x lo6 cells per millilitre in RPMI-SM (approximately a 0.1 % suspension).

+

Luminometric assay

A stock solution of 1 x lo-' mol/l luminol (Sigma Chemical Co, Poole, UK) was prepared in DMSO. M luFrom this a working solution of 4 x minol was made up in RPMI-SM. The mononuc-

245

A CHEMILUMINESCENCE ASSAY FOR ERYTHROPHAGOCYTOSIS

lear leukocytes were taken from liquid nitrogen storage and thawed rapidly in a waterbath at 37 "C. The cells were transferred to tubes containing RPMI 1640 + 10 % FCS, pH 7.5 and centrifuged at 500 g for 5 minutes. The cells were then resuspended in RPMI-SM, pH 8.0. 3 x lo6 cells in 325 p1 RPMI-SM were put into Clinicon cuvettes (Pharmacia-LKB, Milton Keynes, UK) and incubated at 37°C for 15 minutes. 1OOpl of luminol working solution was then added to give a final concentration of 8 x lo-' mol/l. This was followed by 75 p1 of RPMI-SM containing 9 x 10' erythrocytes and the cuvettes inserted into the carousel, thermostatically controlled at 37"C, of an LKB 1251 luminometer linked to an Apple IIe running the LKB 1251-124 Phagoprogram. Continuous light was monitored for 120 minutes with mixing on the first cycle only. The area under the curve of light output for sensitized erythrocytes was divided by that for unsensitized control erythrocytes to give an opsonic index. The ddccee phenotype erythrocytes served as a check on the specificity of the antibody. For each parameter investigated mononuclear leukocytes from a single donor were used. But the same single-donor mononuclear leukocytes were not used for all parameters.

Phagocytic assay

The phagocytic assay is a modification of that of Schanfield et al. (1980). Briefly, 0.5 x lo6 thawed mononuclear leukocytes were added to each chamber of a Labtek tissue culture slide (Miles Laboratories, Slough, UK). The monocytes in RPMI 1640 were adhered at 37°C in a 5 % CO, atmosphere for 30 minutes. The non-adherent cells were aspirated, the medium replaced with 250 p1 RPMI 1640 10% FCS and the monocytes rested for 30 minutes at 37 "C in a 5 % CO, atmosphere. 100 p1 of a 0.1 % suspension of erythrocytes were added and incubated for 60 minutes as above. After incubation the non-adhered cells were aspirated and the plastic wells removed for staining. Staining was completed and assays were evaluated by finding the percentage phagocytosis which is derived by counting 200 monocytes. Those monocytes containing at least one erythrocyte or to which fragments of erythrocytes were adhering, were scored as positive and the formula given by

Hunt et al. (1982) was applied. Percentage phagocytosis -

No. positive monocytes x 100 200

R ESULTS

The thawed mononuclear leukocytes consisted of 44.6 f 3.1 % (mean f SD) monocytes (six preparations). Recovery was 72.2 f 18.8% with 88.0 f 1.4% being viable (mean f SD). There were no polymorphonuclear cells seen in the stained preparation of the cells. The recovered mononuclear leukocytes responded to IgG sensitized erythrocytes by phagocytosing the erythrocytes and undergoing a respiratory burst, detected as light emitted by the luminometer. A typical response shown in Fig. 1. The peak chemiluminescent response was typically seen between 35 and 45 minutes after

50

r

40

-

00 00 0 0 0

O0 0

0

0

- 30 ->E

0

-

.

:..I

.-sE

W

O-

0

0 0

0

0

0 0

0

0

0 0

,"

oo~ooo

+

0

20

40

60

80

100

120

Timefmin)

Figure 1. Chemiluminescence time course of (a) mononuclear leukocytes stimulated with (i) anti-D IgG sensitized erythrocytes, phenotype DCcEe ( O ) ,(ii) anti-D IgG sensit(iii) unsensitized ized erythrocyte, phenotype ddccee (0). erythrocytes, phenotype DCcEe (e),and (b) non-adherent mononuclear leukocytes stimulated with anti-D IgG sensitized erythrocytes, phenotype DCcEe (*). Each point is the mean of three observations.

246

I. DOWNING, J. G. TEMPLETON, R. MITCHELL AND R. H. FRASER

initiation of the assay. After 120 minutes the response had returned to control levels. It was found that continual mixing depressed the chemiluminescent response; however, an initial mix of eight seconds enhanced the response and made it more reproducible (data not shown). We also demonstrated that, if the adherent mononuclear leukocytes were depleted (adhered as for the phagocytic assay) and the chemiluminescent response of the non-adherent cell population compared to unfractionated cells, then the response decreased to below the level of chemiluminescence seen with unfractionated leukocytes and unsensitized erythrocytes (Fig. 1). The concentration of luminol was found to affect the chemiluminescent response. The optimum was found to be 8 x 10-5mol/l with light emission marginally decreasing at concentrations above this (Fig. 2). This could possibly be due to the increased concentration of DMSO used to initially solubilize the luminol as postulated by Easmon et al. (1980). However, when this experiment was repeated, dissolving luminol in 0.1 mol/l NaOH, the chemiluminescent response plateaued at 8 x lo-’ mol/l luminol. Thus it may, more likely, be an indication that the luminol is in excess above that concentration. We also found that the chemiluminescent response plateaued or decreased slightly if more 9 -

--

8 X W

z 7 H

---

--

u H z

g 6 a 0

5 4

--c

t

,

1

1

1

1

1

1

1~ 1

1

I

7 -

X

w

a Z

6 : . -

-

- 5 -

.

v

*

H

.

54:

r n . a

0

. .

3 :

E 0 No.

1

2

3

MONONUCLEAR L E U K O C Y T E S ( X 1 0

4 6

)

Figure 3. The effect of mononuclear leukocyte numbers on their chemiluminescent response to stimulation by 9 x lo5 erythrocytes sensitized with anti-D IgG. Each point is the f SEM of three observations

than 3 x lo6 mononuclear leukocytes were used (Fig. 3). The quantity of erythrocytes affected the chemiluminescent response markedly (Fig. 4). This is probably because haemoglobin may interfere with the luminol reaction or may absorb the emitted light. Phenol red also absorbs in this range; hence our choice of a phenol red-free medium for the assay. The phenotype of the erythrocytes only marginally affected the chemiluminescence assay, as long as the D antigen was present on the red cell. Conversely if the D antigen was not present (e.g. phenotype ddccee) the response was abolished (Table 1). We found the pH of the medium used in the chemiluminescence assay to be important. As shown in Fig. 5 the optimal pH of the mononuclear leukocytes at the start of the assay was pH 8.0, any variation from this pH adversely affecting the chemiluminescent response. We also found in parallel experiments that the pH of the media rose markedly. This seemed to be due to simple loss of CO, from the media, since the pH was maintained by a simple HCO;/H,CO, system, with CO, usually supplied as 5 % of the atmosphere, not possible when using the luminometer. In experiments using samples with and without mononuclear leukocytes, with and without erythrocytes, and without either cell type the pH rose at similar rates. This pH rise was also independent of whether the mononuclear leukocytes were engaged in erythro-

247

A CHEMILUMINESCENCE ASSAY FOR ERYTHROPHAGOCYTOSIS 7 r

6.5 6.0

x

5.5

-

w

n

z

5.0 1

0

4.5

:

4.0

-

w

g (/i

a

0

3.5

-

c

3.0

2

o

2 NO.

4

6

8

1

0

1

2

ERYTHROCYTES ( x 105 )

6.5

7.0

7.5

8.0

8.5

PH

Figure 4. The effect of numbers of IgG sensitized erythrocytes on the chemiluminescent response of 3 x 1 O6 monoSEM of three nuclear leukocytes. Each point in the mean observations.

*

Table 1. The effect of the chemiluminescent assay of erythrocyte Rh phenotype. Erythrocyte Rh phenotype

Opsonic index (fSEMI

DCcEe DccEE DCCee DCcee DccEe ddccee

8.05f 0.30 8.50f 0.32 7.43f 0.78 7.305 0.52 7.46 0.57 1.07f 0.02

*

Each observation is the mean of at least 12 determinations. DCcEe and ddccee were done significantly more often as they were the positive and negative controls respectively. The mononuclear leukocytes used were all from one preparation.

cyte destruction. We tried several buffers to stabilize the pH, such as phosphate, HEPES, bis-tris propane, tris (hydroxymethyl) methylaminopropane suplhonic acid and borate. However, when used in concentrations able to maintain the pH (25-50 mmol/l) they inhibited the chemiluminescent response. The best tolerated was 5 mmol/l phosphate, with virtually no effect on the chemiluminescence, and therefore, although its buffering effect was marginal, we chose RPMI 1640 Searle modification which incorporates approximately

Figure 5. Chemiluminescent response of 3 x lo6 mononuclear leukocytes and 9 x lo5 anti-D sensitized erythrocytes and the effect of the initial pH of the culture media used to resuspend them immediately prior to the chemiluminescence assay. Each point is the mean f SEM of three observations

5 mmol/l phosphate buffer in its formulation. This meant that under assay conditions with an initial pH of 8.0, the pH would rise between 0.3 to 0.5 of a pH unit over a 2-hour experiment. Using a 1/50 dilution (5.25 IU) of anti-D immunoglobulin to sensitize the erythrocytes, an intra-assay CV of 5.03% was obtained while the inter-assay CV was 12.92% using two different batches of mononuclear leukocytes for each measurement and different DCcEe phenotype erythrocytes. It was found that the quantity of anti-D immunoglobulin used to sensitize the erythrocytes and hence the amount of immunoglobulin bound on the erythrocyte was reflected in both the chemiluminescent response and in the phagocytic response, measured microscopically. Although it is difficult to compare such dissimilar quantitations, one open-ended, the other fixed at 100 %, we have taken the percentage difference of the chemiluminescent response of sensitized erythrocytes compared to unsensitized erythrocytes when exposed to monocytes, so that zero is equivalent to the chemiluminescent response of unsensitized erythrocytes. These values have been compared with the percentage phagocytosis (Fig. 6) with the slope of the line giving an indication of the rate of change of the 'signal' and thus an indirect indication of the sensi-

248

I. DOWNING, J. G. TEMPLETON, R. MITCHELL AND R. H. FRASER m v)

C 0

nu, u , m m e E x 0

U 0

.-I k

h r

c c o x E k o w C .rl

u

E O 3 u ,

J .rl

c

Q-

.-I

o w

C

m m

400

t

i

o m C

0 k

0 c

0

-u

Q - m

u, .?I

.-I v) .rl

0

m u , 010

c

0 .-I

mu, o c c m cu, m c

I

+- .

/

0 3

I

a + n o ~

0.05

0.1

0.5

1.0

o c c 0 u k m n

c x

u m

0 0

r

a

~~-

5.0

10.0

Quontity o f Anti-D Immunoglobulin Used t o S e n s i t i s e Erythrocytes Figure 6. The effect of the concentration of the anti-D IgG used to sensitize the erythrocyte on the chemilurninescent

assay ( 0 )and the microscopic assay SEM of five experiments. mean

tivity. From this it can be seen that the overall sensitivities of the two assays are broadly similar. However, if twice the standard deviation of the control unsensitized erythrocytes was taken as the limit of detection, then the detection limit for the chemiluminescence assay was 0.066 IU anti-D IgG used to sensitize 1 ml of 1 % erythrocyted suspension, while the limit of the phagocytic assay was 0.131 IU anti-D IgG used to sensitize 1 ml of 1 % erythrocyte suspension. In addition, when an antiD IgG concentration of 5.25 IU was used to sensitize the erythrocytes the intra-assay CV for the chemiluminescence assay was much better than that for the phagocytic assay, 5.03% and 10.4% respectively. This reflects the tighter replication of the chemiluminescence assay. The biphasic nature of the curve shown in Fig. 6 for chemiluminescence response over a range of anti-D IgG values used to sensitize erythrocytes is probably due to a complex interplay of the different interactions of the differing concentrations of IgG anti-D isotypes present in the polyclonal antiserum with the two Fcy receptors present on the mononuclear leukocytes under study (Dougherty et al., 1987; Van De Winkel et al., 1989; Zupanska et al., 1985).

DISCUSSION

(0). Each point is the

The use of frozen mononuclear leukocytes obviates their time-consuming preparation prior to each experiment and allows an antibody from a patient to be examined over a considerable time span using the same batch of mononuclear cells. This avoids the donor variation which is inevitably seen (Munn and Chaplin, 1977). It has already been shown (Hunt et al., 1981; Shah et al., 1984) that virtually all monocytes are recovered from cryopreservation, cells lost being entirely lymphocytes. We certainly found the cryopreservation step beneficial, in that it increased the concentration of monocytes in a batch by approximately 10%. Additionally we have seen no loss of activity in mononuclear leukocytes recovered from liquid nitrogen tested at intervals over a 12-month period. We found, as did Hadley and Holburn (1984) and Jungi and Peterhans (1988), that the non-adherent (largely lymphocyte) component of the mononuclear cell preparation did not contribute to the chemiluminescent response, that response residing with the adherent cell population which consisted almost entirely of monocytes. We could not detect any granulocytes

249

A CHEMILUMINESCENCE ASSAY FOR ERYTHROPHAGOCYTOSIS

in our cell preparation; however granulocytes which did avoid separation from the mononuclear cell would be highly unlikely to be viable on thawing (Roos and Boer, 1986) and their chemiluminescent response would be markedly reduced (Boonlayangoor et al., 1980). The curious pH optimum of the chemiluminescence assay, pH 8.0, may be explained as a compromise between the optimum pH for the mononuclear leukocytes, approximately pH 7.4, and the optimum for luminol-generated chemiluminescence with reactive oxygen species, which is near pH 12 (Lee and Seliger, 1972; Gyllenhammar, 1989). The finding that several buffers markedly decreased the chemiluminescent response is not suprising, since Easmon et al. (1980) also noted this effect with HEPES. Cells vary in their sensitivity to buffers, especially 'Good' buffers (Good et al., 1966) and may exhibit toxic reactions at concentrations well tolerated by other cell types. It is also possible that the buffers may interfere directly with the complex luminol reaction. Existing methods for the determination of Fcy receptor dependent events, such as that induced by IgG sensitized erythrocytes, have been quantified by the use Cr-labelled erythrocytes (Walker, 1977; Silvergleid et al, 1978; Newman et al., 1980; Cooper et al., 1984) or phagocytic and rosette microscopic assays (Schanfield et al., 1981; Douglas et al., 1985; Nance et al., 1987). The radioisotopic method has the advantage of sensitivity and reproducibility but has all the problems associated with the handling, storage and disposal of radioactive material and is not always practical in a clinical situation. The phagocytic assay requires intensive microscopic observation and can be open to subjective error. The chemiluminescence assay, while not as sensitive as the "Cr assay, has the benefits of being an automated system. It is faster, simpler to carry out, free from subjective errors and slightly more sensitive than the microscopic assay. The chemiluminescence assay also requires minimum manipulation of the-mononuclear leukocytes, gives closer replicates and is more reproducible than the phagocytic assay. The microscopic assay has already been used to predict the clinical significance of patient-derived erythrocyte antibodies (Schanfield et al., 1981; Nance et al., 1987). The chemiluminescence assay should prove of more utility in such a role being faster and not requiring a skilled technician. Additionally if sensitivity should be a drawback it should be possible to boost it using a variety of

cytokines such as interleukin 1 and 2, platelet activating factor, interferon-gamma and others (Wiener and Garner, 1987; Malkovsky et al., 1987; Onoraki et al., 1985; Valone et al., 1988; Philip and Epstein, 1986).

Acknowledgements O u r thanks are due to the donors who so readily gave of their time, t o our donor staff for carrying o u t the apheresis a n d t o Mrs Anne Howieson for secretarial help.

REFERENCES Absolom, D. R. (1986). Basic methods for the study of phagocytosis. Meth. Enzymol., 132,95- 180. Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F. and Millhouse, E. W. (1980). Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure. Blood, 56, 237-245. Beryum, A. (1983). Isolation of human blood monocytes with Nycodenz, a new non-ionic iodonated gradient medium. Scand. J . Immunol., 11, 429-436. Cooper, P. H., Mayer, P. and Baggioloni, M. (1984). Stimulation of phagocytosis in bone marrow-derived mouse macrophages by bacterial lipopolysaccharide: correlation with biochemical and functional parameters. J . Immunol., 133, 9 13-922. Dougherty, G. J., Selvendram, Y., Murdoch, S., Palmer, D. G. and Hogg, N. (1987). The human mononuclear phagocytic high-affinity Fc receptor, FcRI defined by a monoclonal antibody, 10.1. Eur. J . Immunol., 17, 1453-1459. Douglas, R., Rowthorne, N. V. and Schnieder, J. V. (1985). Some quantitative aspects of the human monocyte erythrophagocytosis and rosette assays. Transfusion, 25, 535-539. Easmon. C. S., Cole. P. J., Williams, A. J. and Hastings, M. (1980). The measurement of opsonic and phagocytic function by luminol-dependent chemiluminescence. Immunology, 41, 67-74. Engelfriet, C. P., Von Dem Bourne, A. E. G. Kr., Beckers, D., Van Der Mueler, F. W., Fleer, A., Roos, D. and Ouweland, W. H. (1981). Immune destruction of red cells. In. A seminar on Immune Mediated Cell Destruction. Bill, C. A. (Ed.), American Association of Blood Banks, Chicago, pp. 93-130. Geffner, J. R., Giordano, M., Serebrinsky, G. and Isturiz, M. (1987). The role of reactive oxygen intermediate in nonspecific monocyte cytotoxicity induced by immune complexes. Clin.E x p . Immunuol., 61, 646-654. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Igawa, S. and Singh, R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry, 5, 467-477. Gyllenhammar, H. (1989). Effects of extracellular pH on neutrophi1 superoxide anion production, and chemiluminescence augmented with luminol, lucigenin or DMNH. J . Clin.Lab. Immunol., 28, 97- 102. Hadley, A. and Holburn, A. M. (1984). The detection of antigranulocyte antibodies by chemiluminescence. Clin. lab. Haematol., 6, 351-361.

250

I. DOWNING, J. G. TEMPLETON, R. MITCHELL AND R. H. FRASER

Hogg, N., Macdonald, S., Slusarenko, M. and Beverley, P. C. L. (1984). Monoclonal antibodies specific for human monocytes, granulocytes and endothelium. Immunology, 53, 753-767. Hunt, J. S., Beck, M. L., Teglmeier, G . E. and Bayer, W. L. (1982). Factors influencing monocyte recognition of human erythrocyte autoantibodies in uitro. Transfusion, 22,355-358. Hunt, S . M., Lionetti, F. J., Valeni, C. R. and Callahan, A. B. (1981). Cryogenic preservation of monocytes from human blood and plateletpheresis cellular residues. Blood, 57, 592-598. Jungi, T. W. and Peterhans, E. (1988). Change in chemiluminescence reactivity pattern during in uitro differentiation of human monocytes to macrophages. Blut, 56, 213-220. Lee, J. and Seliger, H. H. (1972). Quantum yields of the luminol chemiluminescencereaction in aqueous and aprotic solvents. Photochem. Photobiol, 15, 227-237. Logue, G. and Rasse, W. F. (1976). Immunologic mechanisms in autoimmune hemolytic disease. Seminars in Haernatology, 13, 277-289. Malkovsky, M., Loveland, B., North, M., Asherson, B. L., Gao, L., Ward, P. and Fiers, W. (1987). Recombinant interleukin2 directly augments the cytotoxicity of human monocytes. Nature, 325, 262 -265. Munn, R. and Chaplin, H. (1977). Rosette formation by sensitised human red cells - effect of source of peripheral leukocyte monolayers. Vox Sang., 33, 129-142. Nance, S. J., Arndt, P. and Garratty, G. (1987). Predicting the clinical significance of red cell alloantibodies using a monocyte mononuclear assay. Transfusion, 27, 449-452. Newman, S. L., Musson, R. A. and Henson, P. M. (1980). Development of functional complement receptors during in uitro maturation of human monocytes into macrophages J . Immunol., 125, 2236-2244. Onoraki, K., Malsushima, K., Klienerman, E. S., Saito, T. and Oppenheim, J. J. (1985). Role of interleukin 1 in promoting monocyte-mediated tumour cytotoxicity. J . Immunol., 135, 314-320. Philip, R. and Epstein, L. B. (1986). Tumour necrosis factor as immunomodulator and mediator of monocyte cytoxicity induced by itself, gamma-interferon and interleukin I. Nature, 323, 86-89. Roos, D. and De Boer, M. (1986). Purification and cryopreservation of phagocytes from human blood. Meth. Enzymol., 132, 225-243. Schanfield, M. S., Schoeppner, S. L. and Stevens, J. 0. (1980). New approaches to detecting clinically significant antibodies in the laboratory. In Zmmunobiology of the Erythrocyte, Sandler, S . G., Nusbacher, J. and Schanfield, M. S. (Eds),

Alan R. Liss, New York, pp. 305-323. Schanfield, M. S., Stevens, J. 0. and Bauman, D. (1981). The detection of clinically significant erythrocyte alloantibodies using a human mononuclear phagocyte assay. Transfusion, 21, 571-576. Shah, V. O., McCarley, D. L. and Weiner, R. S. (1984). Antibody dependent cell mediated cytotoxicity of cryopreserved human monocytes. Cryobiology, 21, 475-479. Silvergleid, A. J., Welles, R. F., Hafleigh, E. B. and Korn, G. (1978). Compatibility testing using chromium labelled red blood cells in crossmatch positive patients. Transfusion, 18, 8-14. Valone, F. H., Philip, R. and Debs, R. J. (1988). Enhanced human monocyte cytotoxicity by platelet-activating factor. Immunology, 64, 715-718. Van Der Mueler, F. M., Van Der Hart, M., Fleer, A,, Von Dem Bourne, A. E. G . Kr., Engelfriet, C. P. and Van Loghem, J. J. (1978). The role of adherence to human mononuclear phagocytes in the destruction of red cells sensitised with noncomplement binding IgG antibodies. Brit. J . Haematol., 38, 541-549. Van De Winkel, J. G . J., Bonnen, G . J. J. C., Janssen, P. L. W., Vlug, A., Hogg, N. and Tax, W. J. M. (1989). Activity of two types of Fc receptors, FcyRI and FcyRII, in human monocyte cytotoxicity to sensitised erythrocytes. Scand. J . Immunol., 29, 23-31. Von Dem Bourne, A. E. G . Kr., Beckers, D. 0.and Engelfriet,C. P. (1977). Mechanisms of red cell destruction mediated by non-complement binding IgG antibodies: the essential role in uiuo of the Fc part of IgG. Brit. J . Haematol., 36,485-493. Walker, W. S. (1977). Mediation of macrophage cytolytic and phagocytic activities by antibodies of different classes and class-specific Fc receptors. J . Immunol., 119, 367-373. Weiner, R. S. (1976). Cryopreservation of lymphocytes for use in in vitro assays of cellular immunity. 1. Immunol. Meth., 10, 49-60. Weiss, S. J. and Lobuglio, A. F. (1982). Phagocyte generated oxygen metabolism and cellular injury. Lab. Invest., 47,5- 18. Weiss, S. J., King, G . W. and Lobuglio, A. F. (1978). Superoxide generation by monocytes and macrophages. Am. J . Haematol., 4, 1-8. Wiener, E. and Garner, S. F. (1987). The use of macrophages stimulated by immune interferon as indicator cells in the mononuclear phagocyte assay. Clin. Lab. Haemntol., 9, 399-408. Zupanska, B., Brojer, E., Maslanka, M. and Hallberg, T. (1985). A comparison between Fc receptors for IgGl and IgG3 on human monocytes and lymphocytes using anti-Rh antibodies. Vox. Sang., 49, 67-76.

A chemiluminescence assay for erythrophagocytosis.

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononu...
637KB Sizes 0 Downloads 0 Views