Scand J Rheumatology 6: 81-86, 1977
THE NITROBLUE TETRAZOLIUM TEST IN JUVENILE RHEUMATOID ARTHRITIS AND THE STIMULATION OF GRANULOCYTES BY PATIENT'S SERA
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M. John and J. Oppermann From the Pediatric Clinic (Head: Prof. L . Weingartner) of Martin-Luther-University Halle- Wittenberg
ABSTRACT. The spontaneous NBT test was used in 57 children with juvenile rheumatoid arthritis (JRA) and another connective tissue diseases. The patients were grouped according to the various manifestations and divided into different stages of activity. The patients with tendosynovitis and unknown arthralgia showed a negative test. Patients with morbus Wissler without exudative joint manifestations were also NBT-negative, to a large degree. By contrast, all manifestations with joint involvement showed a high per centage of positive results in the NBT test. In a second investigation, the granulocytes of normal persons were stimulated by sera of patients with various manifestations of JRA. The sera of patients with joint manifestations all caused a stimulation of the granulocytes of normal persons, compared with the controls, in which pooled human serum was employed. Stimulation of granulocytes with serum of Wissler patients proved impossible, however.
ation of bacterial infections from other febrile diseases. Today, numerous diseases are known, which produce false-positive or false-negative results in the NBT test (16, 29). The investigations should reveal the functional condition of the granulocytes in patients with various types of juvenile rheumatoid arthritis (JRA). As JRA belongs to the group of diseases with probable immune pathogenesis, and immune- or protein complexes ( 1 3 , 3 2 , 3 3 , 35) could be demonstrated in various tissues and body fluids, we tried to stimulate the granulocytes of normal persons with the sera of patients suffering from various types of JRA.
The NBT test was introduced for the diagnosis of chronic granulamatous disease (1). In this test the yellow dye nitroblue tetrazolium (NBT) in phagocytes has been replaced by the insoluble blue violet formazan. However, this histochemical reaction only occurs when the cell membrane of granulocytes and monocytes. which does not allow NBT to pass through, is altered by phagocytosis of animate or inanimate particles (2, 5, 21, 28. 34), immune complexes (14.23) or other substances (25,30). The NBT is brought to the cells by phagocytosis and is reduced by diaphorase of the phagosomes (9). Thus, with the NBT test it is possible to determine the existing phagocytosis capacity of cells, i.e. the functional condition of the cellular immunity and, furthermore, to gain an insight into the phagocytotic efficiency of granulocytes. The test achieved clinical importance following its introduction by Park et a). (26) for the differenti-
I . The spontaneous test The NBT test was performed according to our own modification (12) of the method of Park et al. (26). 0.1 ml capillary blood was aspirated with a pipette containing I IU heparin. The blood was transferred to a silicated slide, formed with concavities, and 0.1 ml of a 0.2% NBT solution (NBT in 0.9% NaCl and 1/15 mol phosphatebuffered saline, pH 7.4 dissolved in equal portions) was added. Incubation was carried out in a moist chamber at a temperature of 37°C for 30 min. At the end of the incubation period the blood was smeared, dried, and stained with a 1 % safranin solution. From each patient, two preparations were made and of each preparation the formazanpositive cells were determined in 100 granulocytes. At the same time, leukocytes were counted, and together with the differential blood count the absolute neutrophils/mm3 of blood as well as the absolute number of NBT-positive cells/mm3 were determined. The results of the investigations are given as mean values, together with their standard deviations. A reaction of &IS% of NBT-positive granulocytes was considered to be a negative result (12). With this method, we examined 57 children (84 tests) with JRA and other connective tissue diseases. There
METHOD
6 - 77 1x69
82
M.John and J . O p p e r m a n n
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
n = 12
n 113
n = 41
n -14
n.4
1 comploto and incomploto Stlll't dirooro, morbus Wirsler with arthritis
adult typos of juvonile rheumatoid arthritis
-
mono and polyorthricular typor of juvonik rhoumatoid arthritis
morbur
Wisrlor
tondorynovittr and unknown arthrolglo
Fig. 1 . Mean values and standard deviations of NBT test
in different manifestations of JRA. n=number of investigations. were 9 patients with morbus Wissler (14 tests), 28 patients with JRA of the mono- or polyarthritic type (41 tests), 8 patients with JRA of the adult type (13 tests), 8 patients with a complete or incomplete Still's disease (12 tests), and 4 patients with unknown arthralgia and tendosynovitis (4 tests). The classification of the disease into the various activity stages was done according to laboratory and chemical criteria, by means of blood sedimentation rate, diphenyl reaction (17), alphh-globulin, and albumin. 2 . Stimulation of granulocytes of normal persons by sera of patients suffering from different varieties of JRA 10 ml venous blood samples from normal persons were
anticoagulated with sodium ethylene diamine tetraacetate (EDTA) 3 mglml of blood, followed by a 90 min sedimentation at 4°C. Finally, the buffy coat was decanted. The standardization of the leukocytes in autologous serum was performed on 8-6-10-6 cells/O. 1 ml. 0.2 ml buffy coat was added to 0.1 ml of fresh serum of patients suffering from various types of JRA, preincubated at 37°C for 25 min, and incubated after adding of 0.1 ml of a 0.2% NBT solution for 30 min. At the end of the incubation period, the cells were centrifugated at room temperature at 500g for 5 min. Smears were prepared and the further treatment and evaluation followed as described in (I). Sera of 8 patients, suffering from differentjoint manifestations of JRA, and 3 sera of patients with morbus Wissler were examined. In the 20 controls, pooled human serum was employed. Scand J Rhectmatology 6
RESULTS In the 57 children examined having various types of JRA and other diseases connective tissues (84 tests), a positive result was obtained in 64 tests (76%). By classifying the tested patients with JRA and other diseases of the connective tissues according to the distinct manifestation, the results given in Fig. 1 and Table I are obtained. Patients with tendosynovitis and unknown arthralgia showed a negative N B T test. A considerable proportion of patients with morbus Wissler but without exudative joint manifestations were NBTnegative. In contrast, all manifestations of joint involvement demonstrated a high percentage of positive results in the N B T test. The classification of o u r 57 patients with confirmed JRA and tendosynovitis into four different stages of activity according to laboratory criteria at the time of examination, yielded the results presented in Fig. 2 and Table 11. The group with activity stage I consists of 3 patients with tendosynovitis or unknown arthralgia of one patient with an adult type of JRA. Stage I1 group consists mainly of patients with mono- and polyarthritis and the adult
The tiitroblire tetrazoliurn test in jirvenile rheumutoid mrthritis
83
Table I. Results ofthe NBT test in the dgferent types ofjuvenile rheumatoid arthritis n=number of patients, .i-=mean value, s=standard deviation
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
% of
No. of tests
formazan-pos. granulocytes (Xfs)
Formazan-pos. granulocytes
No. of pats. with pos. tests
Diagnosis
n
Tendosynovitis and unknown arthralgia Morbus Wissler Complete and incomplete Still’s disease Adult types of juvenile rheumatoid arthritis Mono- and polyarthritic types of juvenile rheumatoid arthritis
4
4
13+ 2(11-16)
S32f I30
9
14
14rt 9 (3-31)
I 103+832
4=44%
8
12
37f23 (8-31)
2 747+ I 763
7=87%
8
13
3Sk20 (9-65)
I502+762
6=7S%
28
41
38k17 (6-76)
1646+593
26=93%
type of JRA. Patients with morbus Wissler, with and without joint involvement, incomplete Still’s disease, as well as with the mono- and polyarthritic type of JRA, comprise the group with activity stage 111. Activity stage IV consists of 3 patients with Wissler and of one patient with an incomplete Still’s disease. The results of the stimulation of granulocytes are shown in Table 111. In the spontaneous NBT test, 18000
;i ~ o o a J
r
nxl.
n =3 0
(ifS)
all types of JRA with joint involvement showed a positive NBT test, compared with the controls, while all but one patient with morbus Wissler had negative results. The sera of patients with joint manifestations all caused a stimulation of the granulocytes of normal persons, compared with the controls, in which pooled human serum was employed. The stimulation was not connected with latex positivity or negativity of the patient’s serum. The n: 19
n.4
loo
i 12ooa ! 9ooc n
2
n
6000
3000 Y C
0
-
I
abrolbte noutrophilr ,
II
111 stage
+-4
IV of activity
formazan positive neutrophils
Fig. 2. Presentation of the relations between manifestation
activity, number of neutrophils, and of the fonnazan positive cells. n=number of patients.
1=2S%,
84
M.John and J . Oppermann
Table 11. Results of formuzan-positive patients (granulocyteslmm3)of blood and absolute positive granulocytes in patients with juvenile rheumatoid arthritis in different activity stages .i=mean value, s =standard deviation % of pats.
Stage of activity
Formazan-neg. pat.
with formazanpos. tests
I I1
3 out of 4 2 out of 30 6 out of 68 3 out of 4
25 93 a 68 25
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111 1v
Granulocytes (mm3of blood) (Xfs) 6 251f1 362 4 5702 1 347 6 914f3 473 11 742+4 078
stimulation of granulocytes with serum of Wissler patients, however, was not possible (Table HI). Negative results occurred too, when the sera of patients with joint involvement were stored at 4°C for 4-16 h.
Formazan-pos. granulocytes (mm3 of blood) (XfS)
9973449 1705f660 1364t985 I 248f560
thritis (excluding the 4 patients with tendosynovitis and those with JRA not unequivocal according to the ARA criteria (3)), have shown that in this disease in a large proportion (more than 15%) of NBT-positive granulocytes can be expected. In our patients, we found in 64 out of 84 determinations (76%) more than 15% NBT-positive granulocytes, and in 30 out of 84 determinations the number of NBT-positive cells was above 30%. From the calculations and from Fig. 1 and Table I it can be seen that the majority of highly positive reactions (more than 30% of NBT-positive cells) occur in patients with prevalent joint manifestations. The mean values of NBT-positive granulo-
DISCUSSION From the literature it is known that positive results can be obtained with the NBT test in patients with rheumatoid arthritis (4, 6, 8, 11, 15, 21, 22, 23). Other authors, however, have had dissimilar results (7, 10, 24, 3 1). Our investigation of 53 children with various types of confirmed juvenile rheumatoid ar-
Table 111. The results of spontaneous NBT test in different types of juvenile rheumatoid arthritis and the stimulation of the granulocytes in normal persons with patient's serum n=number of investigations, - = not investigated % of formazan% of formazan-
Patient1 agelsex Hi. 191? Fu.11316 Bo.17ld Th.11 2/? Ju .I716 He.112/? Si./7/? He./ 13/6 Sch./l2/d Ab.llZ/d Sch./S/ d Controls
Diagnosis Incomplete Still's disease Adult type of JRA Polyarthritic type of JRA Polyarthritic type of JRA Morbus Wissler with arthritis Adult type of JRA Polyarthritic type of JRA Adult type of JRA Morbus Wissler Morbus Wissler Morbus Wissler
positive granulocytes after stimulation with patient's serum Qfs)
Hyland test
n
positive granulocytes in the spontaneous test Mfs)
Neg.
Neg.
2
18f 3
12 +2
Neg. Pos.
-
2
33f10 25
12 f 3 5.5f2
Neg.
Neg.
0
-
Pos.
Neg.
Neg
I
46
5.5f 1
Neg. Neg.
Neg. Neg.
-
2 1
30f 8
5.5+2 5 f0
Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg.
Neg. Neg. Neg.
I
51 20 2 11f 2 6f 5
C-reactive Latex protein test Pos.
-
1 : 1280 I
-
1 1
3 20
n
6
34
2 2 2 20
8 +5
5 f3 2.5f 1 2.5f 1 3 f l
3.2f1.8
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The nitroblue tetrazolium test in juvenile rheumatoid arthritis
cytes were significantly lower in morbus Wissler and in cases of incomplete and complete Still’s disease. We found similar results in patients with unknown arthralgia and tendosynovitis. Fig. 2 and Table I1 demonstrate that in patients with JRA, of activity stage 11, the highest values of NBT-positive granulocytes were found both in percent (93%) and absolutely (1 705f660 cells). From this, however, we are unable to establish any correlation between the proportion of NBT-positive granulocytes and the inflammatory activity of the rheumatic process. Whereas patients with joint manifestations correspond mainly to activity stage 11, patients with morbus Wissler and incomplete and complete Still’s disease were grouped mainly under activity stages 111 and IV. Unknown arthralgia and children with tendosynovitis correspond to activity stage I. Considering that it is the clinical manifestation and not the activity degree that is important for the result of the NBT test, the question then arises as to the activating mechanisms in these types of manifestation. I t is known that stimulation of the NBT reaction in the granulocytes is possible by means of endotoxin (21, 25), bacteria ( 5 , 21), Candida albicans (28), Latex particles (2, 34) zymosan (l8), immune complexes (14,23), NBT heparin fibrinogen complexes, and other substances (27, 30). The present concept of the etiopathogenesis of JRA is that the bacterial and viral noxes (19, 20), and their resulting immunological reactions with formation of immune complexes and autoantibody could be of decisive importance. Pachmann et al. (23) were able to demonstrate that it is possible to stimulate the phagocytes with the rheumafactor of positive and negative sera of patients suffering from rheumatoid arthritis. Our results show that even in mainly seronegative JRA, a stimulation of the neutrophil granulocytes is successful. These results could not be obtained by employing sera of patients with exclusively visceral types of JRA and symptoms of Wi ssl er. It is known that in patients with rheumatoid arthritis, immune complexes are found not only in synovial fluid and synovial membrane (32) but also in the peripheral circulation (33). These immune complexes are subjected to phagocytosis in vitro by granulocytes and cells of the synovial fluid (13, 35). The stimulation of granulocytes in the NBT test with sera of JRA patients having joint manifestations, as demonstrated by us, indicates that at least
85
in these manifestations of JRA, immune complexes are present in serum. These, the NBT reaction activating complexes, proved to be highly unstable, as storage even at 4°C results in inactivation. The failure to stimulate neutrophils with serum of patients suffering from exclusively visceral types of JRA and morbus Wissler could mean that the formation of immune complexes or humoral immune mechanisms, at least primary or transitory, is of no decisive importance.
REFERENCES I . Baehner, R. L. & Nathan, D. G.: Leukocyte oxidase: defective activity in chronic granulomatous disease. Science 155:835, 1967. 2. Baehner, R. L. & Nathan, D. G.: Quantitative nitroblue tetrazolium test in chronic granulomatous disease. New Engl J Med 278: 971, 1968. 3. Bass, J. C., Cassidy, J. T., Duran, B. S., Fink, C. W., Jacobs, J. C., Markowitz, M., Reynolds, W. E., Schaller, J., Stillmann, J. S. &Wallace, S. L.: Criteria for the classification of juvenile rheumatoid arthritis. Bull Rheum Dis23:712, 1972/73. 4. Bittner, J. S., Kieff, E., Windhorst, D. & Meier, P.: The use of the unstimulated nitroblue tetrazolium test as a routine screening test for bacterial infection in an adult population: a reassessment. Am J Clin Pathol 60: 843, 1973. 5 . Cocchi, P., Mori, S. & Becattini, A.: II test al nitroblue tetrazolium nel prematuro. Minerva Pediatr 23: 185, 1971. 6. Farhadian, H.: The use of NBT test in rheumatic diseases in childhood. Proc Inst Med Chicago28: 471, 1971. 7. Feigin, R. D., Shackelford, P. S., Choi, S. C., Flake, K. K., Franklin, F. A. & Eisenberg, C. S.: Nitroblue tetrazolium dye test as an aid in the differential diagnosis of febrile disorders. J Pediatr 78: 230, 1971. 8. Fine, D. P., Morney, S. R. J., Colley, D. G., Sergent, J. S. & DesPrez, R. M.: Effect of divalent ion chelators on zymosan decomplementation of human serum. Fed Proc3I: 787, 1972. 9. Holmes, B. & Good, R. A.: Metabolic and functional abnormalities of human neutrophils. In Phagocytic and mechanisms in health and disease (ed. R. C. Williams and H. H. Fudenberg), p. 51. Int. Med. Book Corp., New York, 1972. 10. Humbert, J. R., Kurtz, M. L. & Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants. Pediatrics45: 125, 1970. 1 1 . John, M. & Oppermann, J.: Der spontane NBT-test in Granulocyten von Patienten mit juveniler Rheumatoid-Arthritis. Z Rheumatol34: 336, 1975. 12. John, M.: Der spontane NBT-test bei gesunden Neugeborenen und Kindern -ein Vergleich zweier Methoden. Dtsch Gesundhetsw30: 1172, 1975. 13. Kinsella, T. D., Baurn, J. & Schiff. M.: Immunofluorescent demonstration of an IgG-1 complex in synovial
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
86
M.John and J . Oppermunn
lining cells of rheumatoid membrane. Clin Exp Immunol4: 265, 1969. 14. Koch, C. & Hoiby, N.: Nitroblue tetrazolium staining of human neutrophil granulocytes. Acta path mikrobiol Scanddl: 787, 1973. 15. Kuduk, I., Pytko, H. & Kowalewski, J.: Test redukcji NBT u chorych na reumatoidalue zapalenie stawow. Reumatologia (Warszawa) 18: 249, 1975. 16. Lace, J. K., Tan, J. S., & Watanakunakorn, Ch.: An appraisal of the nitroblue tetrazolium reduction test. Am J Med 58:685, 1975. 17. Metzke, H. & Oppermann, J.: Die Diphenylaminreaktion als Aktivitatsparameter bei der juvenilen Rheumatoid-Arthritis. Dtsch Gesundheitsw 30: 1857, 1975. 18. Nathan, D. G., Baehner, R. L. & Waever, D. K.: Failure of nitroblue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest48: 1895, 1%9. 19. Nowoslawski, A. & Brzosko, W. J.: Immunopathology of rheumatoid arthritis. The rheumatoid synovitis. Pathol Eur2: 198, 1%7. 20. Nowoslawski, A. & Brzosko, W. J.: Immunpathology of rheumatoid arthritis. 11. rheumatoid nodule (the rheumatoid granuloma). Pathol Eur 2: 302, 1%7. 21. Nydegger, U . E., Miescher, A,, Anner, R. M., Creighton, D. W., Lambert, P. H. & Miescher, P. A.: Serum and cellular factor involvement in nitroblue tetrazolium (NBT) reduction by human neutrophils. Klin Wochenschr51: 377, 1973. 22. Okuda, K., Tanokoro, I. & Sekido, M.: The NBT test in Behcet’s syndrome. New Engl J Med 290: 915, 1974. 23. Pachmann, L. M., Jayanetra, P. & Rothberg. R. M.: Rheumatoid sera and soluble complexes: Nitroblue tetrazolium dye test and hexose monophosphate shunt activation. Pediatrics 52: 823, 1973. 24. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid. J Pediatrics 78: 376, 1971. 25. Park, B. H. & Good, R. A.: NBT test stimulated. Lancet 2: 616, 1970. 26. Park, B. H., Fikrig, S. M.& Smithwick, E. M.: Infection and nitroblue tetrazolium reduction by neutrophils. A diagnostic aid. Lancet 2: 532, 1%8. 27. Phelps, P.: Polymorphonuclear leukocyte motility in
Srunil J Rheumnrulogy 6
28.
29. 30. 31.
32.
33.
34.
35.
vitro. 111. Possible release of a chemotactic substance following phagocytosis of urate crystals by polymorphonuclear leukocytes. Arthritis Rheum 12: 197, 1969. Preisig, E. & Hitzig. W. H., Nitroblue-tetrazolium test for the detection of chronic granulomatous disease. Technical modification. Eur J Clin Invest 1:409. 1971. Segal, A. W.: Nitroblue-tetrazolium test. Lancet 2: 1248, 1974. Segal, A. W. & Levi, A. J.: The mechanism of entry of dye into neutrophils in the nitroblue tetrazolium (NBT)test. Clin Sci Mol Med45:817, 1973. Wenger, M. E. & Bole, G. G.: Nitroblue tetrazolium dye reduction by peripheral leukocytes from rheumatoid arthritis and systemic lupus erythematosus patients measured by a histochemical and spectrophotometric method. J Lab Clin Med82:513, 1973. Winchester, R. J . , Agnello, E. & Kunkel, H. G.: Gammaglobulin complexes in synovial fluids of patients with rheumatoid arthritis: partial characterization and relationship to lowered complement levels. Clin Exp lmmunol6: 689, 1970. Winchester, R. J., Liturin, S. D., Koftler, D. & Kunkel, H. G.: Arthritis Rheum 14:650, 1971. Ref.: Eibl, M.: Nachweis der cellularen Immunitat gegen homologes und autologes, aggregiertes sowie gegen komplexgebundenes IgG bei primar chronischer Polyarthritis. Z Immunitaetsforsch Allerg Klin Immunol 144: 103, 1972. Windhorst, D. B., Holmes, B. & Good, R. A.: A newly defined x-linked trait in man with demonstration of the lyon effection carrier females. Lancet I: 737, 1%7. Zucker-Franklin, D.: The phagosomes in rheumatoid synovial fluid leukocytes: A light fluorescence and electron microscope study. Arthritis Rheum 9: 24, 1966.
Submitted for publication August 19, 1976
M. John Pediatric Clinic Martin-Luther-University Halle-Wittenberg
THE NITROBLUE TETRAZOLIUM TEST IN JUVENILE RHEUMATOID ARTHRITIS AND THE STIMULATION OF GRANULOCYTES BY PATIENT'S SERA
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
M. John and J. Oppermann From the Pediatric Clinic (Head: Prof. L . Weingartner) of Martin-Luther-University Halle- Wittenberg
ABSTRACT. The spontaneous NBT test was used in 57 children with juvenile rheumatoid arthritis (JRA) and another connective tissue diseases. The patients were grouped according to the various manifestations and divided into different stages of activity. The patients with tendosynovitis and unknown arthralgia showed a negative test. Patients with morbus Wissler without exudative joint manifestations were also NBT-negative, to a large degree. By contrast, all manifestations with joint involvement showed a high per centage of positive results in the NBT test. In a second investigation, the granulocytes of normal persons were stimulated by sera of patients with various manifestations of JRA. The sera of patients with joint manifestations all caused a stimulation of the granulocytes of normal persons, compared with the controls, in which pooled human serum was employed. Stimulation of granulocytes with serum of Wissler patients proved impossible, however.
ation of bacterial infections from other febrile diseases. Today, numerous diseases are known, which produce false-positive or false-negative results in the NBT test (16, 29). The investigations should reveal the functional condition of the granulocytes in patients with various types of juvenile rheumatoid arthritis (JRA). As JRA belongs to the group of diseases with probable immune pathogenesis, and immune- or protein complexes ( 1 3 , 3 2 , 3 3 , 35) could be demonstrated in various tissues and body fluids, we tried to stimulate the granulocytes of normal persons with the sera of patients suffering from various types of JRA.
The NBT test was introduced for the diagnosis of chronic granulamatous disease (1). In this test the yellow dye nitroblue tetrazolium (NBT) in phagocytes has been replaced by the insoluble blue violet formazan. However, this histochemical reaction only occurs when the cell membrane of granulocytes and monocytes. which does not allow NBT to pass through, is altered by phagocytosis of animate or inanimate particles (2, 5, 21, 28. 34), immune complexes (14.23) or other substances (25,30). The NBT is brought to the cells by phagocytosis and is reduced by diaphorase of the phagosomes (9). Thus, with the NBT test it is possible to determine the existing phagocytosis capacity of cells, i.e. the functional condition of the cellular immunity and, furthermore, to gain an insight into the phagocytotic efficiency of granulocytes. The test achieved clinical importance following its introduction by Park et a). (26) for the differenti-
I . The spontaneous test The NBT test was performed according to our own modification (12) of the method of Park et al. (26). 0.1 ml capillary blood was aspirated with a pipette containing I IU heparin. The blood was transferred to a silicated slide, formed with concavities, and 0.1 ml of a 0.2% NBT solution (NBT in 0.9% NaCl and 1/15 mol phosphatebuffered saline, pH 7.4 dissolved in equal portions) was added. Incubation was carried out in a moist chamber at a temperature of 37°C for 30 min. At the end of the incubation period the blood was smeared, dried, and stained with a 1 % safranin solution. From each patient, two preparations were made and of each preparation the formazanpositive cells were determined in 100 granulocytes. At the same time, leukocytes were counted, and together with the differential blood count the absolute neutrophils/mm3 of blood as well as the absolute number of NBT-positive cells/mm3 were determined. The results of the investigations are given as mean values, together with their standard deviations. A reaction of &IS% of NBT-positive granulocytes was considered to be a negative result (12). With this method, we examined 57 children (84 tests) with JRA and other connective tissue diseases. There
METHOD
6 - 77 1x69
82
M.John and J . O p p e r m a n n
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
n = 12
n 113
n = 41
n -14
n.4
1 comploto and incomploto Stlll't dirooro, morbus Wirsler with arthritis
adult typos of juvonile rheumatoid arthritis
-
mono and polyorthricular typor of juvonik rhoumatoid arthritis
morbur
Wisrlor
tondorynovittr and unknown arthrolglo
Fig. 1 . Mean values and standard deviations of NBT test
in different manifestations of JRA. n=number of investigations. were 9 patients with morbus Wissler (14 tests), 28 patients with JRA of the mono- or polyarthritic type (41 tests), 8 patients with JRA of the adult type (13 tests), 8 patients with a complete or incomplete Still's disease (12 tests), and 4 patients with unknown arthralgia and tendosynovitis (4 tests). The classification of the disease into the various activity stages was done according to laboratory and chemical criteria, by means of blood sedimentation rate, diphenyl reaction (17), alphh-globulin, and albumin. 2 . Stimulation of granulocytes of normal persons by sera of patients suffering from different varieties of JRA 10 ml venous blood samples from normal persons were
anticoagulated with sodium ethylene diamine tetraacetate (EDTA) 3 mglml of blood, followed by a 90 min sedimentation at 4°C. Finally, the buffy coat was decanted. The standardization of the leukocytes in autologous serum was performed on 8-6-10-6 cells/O. 1 ml. 0.2 ml buffy coat was added to 0.1 ml of fresh serum of patients suffering from various types of JRA, preincubated at 37°C for 25 min, and incubated after adding of 0.1 ml of a 0.2% NBT solution for 30 min. At the end of the incubation period, the cells were centrifugated at room temperature at 500g for 5 min. Smears were prepared and the further treatment and evaluation followed as described in (I). Sera of 8 patients, suffering from differentjoint manifestations of JRA, and 3 sera of patients with morbus Wissler were examined. In the 20 controls, pooled human serum was employed. Scand J Rhectmatology 6
RESULTS In the 57 children examined having various types of JRA and other diseases connective tissues (84 tests), a positive result was obtained in 64 tests (76%). By classifying the tested patients with JRA and other diseases of the connective tissues according to the distinct manifestation, the results given in Fig. 1 and Table I are obtained. Patients with tendosynovitis and unknown arthralgia showed a negative N B T test. A considerable proportion of patients with morbus Wissler but without exudative joint manifestations were NBTnegative. In contrast, all manifestations of joint involvement demonstrated a high percentage of positive results in the N B T test. The classification of o u r 57 patients with confirmed JRA and tendosynovitis into four different stages of activity according to laboratory criteria at the time of examination, yielded the results presented in Fig. 2 and Table 11. The group with activity stage I consists of 3 patients with tendosynovitis or unknown arthralgia of one patient with an adult type of JRA. Stage I1 group consists mainly of patients with mono- and polyarthritis and the adult
The tiitroblire tetrazoliurn test in jirvenile rheumutoid mrthritis
83
Table I. Results ofthe NBT test in the dgferent types ofjuvenile rheumatoid arthritis n=number of patients, .i-=mean value, s=standard deviation
Scand J Rheumatol Downloaded from informahealthcare.com by University of British Columbia on 12/11/14 For personal use only.
% of
No. of tests
formazan-pos. granulocytes (Xfs)
Formazan-pos. granulocytes
No. of pats. with pos. tests
Diagnosis
n
Tendosynovitis and unknown arthralgia Morbus Wissler Complete and incomplete Still’s disease Adult types of juvenile rheumatoid arthritis Mono- and polyarthritic types of juvenile rheumatoid arthritis
4
4
13+ 2(11-16)
S32f I30
9
14
14rt 9 (3-31)
I 103+832
4=44%
8
12
37f23 (8-31)
2 747+ I 763
7=87%
8
13
3Sk20 (9-65)
I502+762
6=7S%
28
41
38k17 (6-76)
1646+593
26=93%
type of JRA. Patients with morbus Wissler, with and without joint involvement, incomplete Still’s disease, as well as with the mono- and polyarthritic type of JRA, comprise the group with activity stage 111. Activity stage IV consists of 3 patients with Wissler and of one patient with an incomplete Still’s disease. The results of the stimulation of granulocytes are shown in Table 111. In the spontaneous NBT test, 18000
;i ~ o o a J
r
nxl.
n =3 0
(ifS)
all types of JRA with joint involvement showed a positive NBT test, compared with the controls, while all but one patient with morbus Wissler had negative results. The sera of patients with joint manifestations all caused a stimulation of the granulocytes of normal persons, compared with the controls, in which pooled human serum was employed. The stimulation was not connected with latex positivity or negativity of the patient’s serum. The n: 19
n.4
loo
i 12ooa ! 9ooc n
2
n
6000
3000 Y C
0
-
I
abrolbte noutrophilr ,
II
111 stage
+-4
IV of activity
formazan positive neutrophils
Fig. 2. Presentation of the relations between manifestation
activity, number of neutrophils, and of the fonnazan positive cells. n=number of patients.
1=2S%,
84
M.John and J . Oppermann
Table 11. Results of formuzan-positive patients (granulocyteslmm3)of blood and absolute positive granulocytes in patients with juvenile rheumatoid arthritis in different activity stages .i=mean value, s =standard deviation % of pats.
Stage of activity
Formazan-neg. pat.
with formazanpos. tests
I I1
3 out of 4 2 out of 30 6 out of 68 3 out of 4
25 93 a 68 25
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111 1v
Granulocytes (mm3of blood) (Xfs) 6 251f1 362 4 5702 1 347 6 914f3 473 11 742+4 078
stimulation of granulocytes with serum of Wissler patients, however, was not possible (Table HI). Negative results occurred too, when the sera of patients with joint involvement were stored at 4°C for 4-16 h.
Formazan-pos. granulocytes (mm3 of blood) (XfS)
9973449 1705f660 1364t985 I 248f560
thritis (excluding the 4 patients with tendosynovitis and those with JRA not unequivocal according to the ARA criteria (3)), have shown that in this disease in a large proportion (more than 15%) of NBT-positive granulocytes can be expected. In our patients, we found in 64 out of 84 determinations (76%) more than 15% NBT-positive granulocytes, and in 30 out of 84 determinations the number of NBT-positive cells was above 30%. From the calculations and from Fig. 1 and Table I it can be seen that the majority of highly positive reactions (more than 30% of NBT-positive cells) occur in patients with prevalent joint manifestations. The mean values of NBT-positive granulo-
DISCUSSION From the literature it is known that positive results can be obtained with the NBT test in patients with rheumatoid arthritis (4, 6, 8, 11, 15, 21, 22, 23). Other authors, however, have had dissimilar results (7, 10, 24, 3 1). Our investigation of 53 children with various types of confirmed juvenile rheumatoid ar-
Table 111. The results of spontaneous NBT test in different types of juvenile rheumatoid arthritis and the stimulation of the granulocytes in normal persons with patient's serum n=number of investigations, - = not investigated % of formazan% of formazan-
Patient1 agelsex Hi. 191? Fu.11316 Bo.17ld Th.11 2/? Ju .I716 He.112/? Si./7/? He./ 13/6 Sch./l2/d Ab.llZ/d Sch./S/ d Controls
Diagnosis Incomplete Still's disease Adult type of JRA Polyarthritic type of JRA Polyarthritic type of JRA Morbus Wissler with arthritis Adult type of JRA Polyarthritic type of JRA Adult type of JRA Morbus Wissler Morbus Wissler Morbus Wissler
positive granulocytes after stimulation with patient's serum Qfs)
Hyland test
n
positive granulocytes in the spontaneous test Mfs)
Neg.
Neg.
2
18f 3
12 +2
Neg. Pos.
-
2
33f10 25
12 f 3 5.5f2
Neg.
Neg.
0
-
Pos.
Neg.
Neg
I
46
5.5f 1
Neg. Neg.
Neg. Neg.
-
2 1
30f 8
5.5+2 5 f0
Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg.
Neg. Neg. Neg.
I
51 20 2 11f 2 6f 5
C-reactive Latex protein test Pos.
-
1 : 1280 I
-
1 1
3 20
n
6
34
2 2 2 20
8 +5
5 f3 2.5f 1 2.5f 1 3 f l
3.2f1.8
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The nitroblue tetrazolium test in juvenile rheumatoid arthritis
cytes were significantly lower in morbus Wissler and in cases of incomplete and complete Still’s disease. We found similar results in patients with unknown arthralgia and tendosynovitis. Fig. 2 and Table I1 demonstrate that in patients with JRA, of activity stage 11, the highest values of NBT-positive granulocytes were found both in percent (93%) and absolutely (1 705f660 cells). From this, however, we are unable to establish any correlation between the proportion of NBT-positive granulocytes and the inflammatory activity of the rheumatic process. Whereas patients with joint manifestations correspond mainly to activity stage 11, patients with morbus Wissler and incomplete and complete Still’s disease were grouped mainly under activity stages 111 and IV. Unknown arthralgia and children with tendosynovitis correspond to activity stage I. Considering that it is the clinical manifestation and not the activity degree that is important for the result of the NBT test, the question then arises as to the activating mechanisms in these types of manifestation. I t is known that stimulation of the NBT reaction in the granulocytes is possible by means of endotoxin (21, 25), bacteria ( 5 , 21), Candida albicans (28), Latex particles (2, 34) zymosan (l8), immune complexes (14,23), NBT heparin fibrinogen complexes, and other substances (27, 30). The present concept of the etiopathogenesis of JRA is that the bacterial and viral noxes (19, 20), and their resulting immunological reactions with formation of immune complexes and autoantibody could be of decisive importance. Pachmann et al. (23) were able to demonstrate that it is possible to stimulate the phagocytes with the rheumafactor of positive and negative sera of patients suffering from rheumatoid arthritis. Our results show that even in mainly seronegative JRA, a stimulation of the neutrophil granulocytes is successful. These results could not be obtained by employing sera of patients with exclusively visceral types of JRA and symptoms of Wi ssl er. It is known that in patients with rheumatoid arthritis, immune complexes are found not only in synovial fluid and synovial membrane (32) but also in the peripheral circulation (33). These immune complexes are subjected to phagocytosis in vitro by granulocytes and cells of the synovial fluid (13, 35). The stimulation of granulocytes in the NBT test with sera of JRA patients having joint manifestations, as demonstrated by us, indicates that at least
85
in these manifestations of JRA, immune complexes are present in serum. These, the NBT reaction activating complexes, proved to be highly unstable, as storage even at 4°C results in inactivation. The failure to stimulate neutrophils with serum of patients suffering from exclusively visceral types of JRA and morbus Wissler could mean that the formation of immune complexes or humoral immune mechanisms, at least primary or transitory, is of no decisive importance.
REFERENCES I . Baehner, R. L. & Nathan, D. G.: Leukocyte oxidase: defective activity in chronic granulomatous disease. Science 155:835, 1967. 2. Baehner, R. L. & Nathan, D. G.: Quantitative nitroblue tetrazolium test in chronic granulomatous disease. New Engl J Med 278: 971, 1968. 3. Bass, J. C., Cassidy, J. T., Duran, B. S., Fink, C. W., Jacobs, J. C., Markowitz, M., Reynolds, W. E., Schaller, J., Stillmann, J. S. &Wallace, S. L.: Criteria for the classification of juvenile rheumatoid arthritis. Bull Rheum Dis23:712, 1972/73. 4. Bittner, J. S., Kieff, E., Windhorst, D. & Meier, P.: The use of the unstimulated nitroblue tetrazolium test as a routine screening test for bacterial infection in an adult population: a reassessment. Am J Clin Pathol 60: 843, 1973. 5 . Cocchi, P., Mori, S. & Becattini, A.: II test al nitroblue tetrazolium nel prematuro. Minerva Pediatr 23: 185, 1971. 6. Farhadian, H.: The use of NBT test in rheumatic diseases in childhood. Proc Inst Med Chicago28: 471, 1971. 7. Feigin, R. D., Shackelford, P. S., Choi, S. C., Flake, K. K., Franklin, F. A. & Eisenberg, C. S.: Nitroblue tetrazolium dye test as an aid in the differential diagnosis of febrile disorders. J Pediatr 78: 230, 1971. 8. Fine, D. P., Morney, S. R. J., Colley, D. G., Sergent, J. S. & DesPrez, R. M.: Effect of divalent ion chelators on zymosan decomplementation of human serum. Fed Proc3I: 787, 1972. 9. Holmes, B. & Good, R. A.: Metabolic and functional abnormalities of human neutrophils. In Phagocytic and mechanisms in health and disease (ed. R. C. Williams and H. H. Fudenberg), p. 51. Int. Med. Book Corp., New York, 1972. 10. Humbert, J. R., Kurtz, M. L. & Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants. Pediatrics45: 125, 1970. 1 1 . John, M. & Oppermann, J.: Der spontane NBT-test in Granulocyten von Patienten mit juveniler Rheumatoid-Arthritis. Z Rheumatol34: 336, 1975. 12. John, M.: Der spontane NBT-test bei gesunden Neugeborenen und Kindern -ein Vergleich zweier Methoden. Dtsch Gesundhetsw30: 1172, 1975. 13. Kinsella, T. D., Baurn, J. & Schiff. M.: Immunofluorescent demonstration of an IgG-1 complex in synovial
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lining cells of rheumatoid membrane. Clin Exp Immunol4: 265, 1969. 14. Koch, C. & Hoiby, N.: Nitroblue tetrazolium staining of human neutrophil granulocytes. Acta path mikrobiol Scanddl: 787, 1973. 15. Kuduk, I., Pytko, H. & Kowalewski, J.: Test redukcji NBT u chorych na reumatoidalue zapalenie stawow. Reumatologia (Warszawa) 18: 249, 1975. 16. Lace, J. K., Tan, J. S., & Watanakunakorn, Ch.: An appraisal of the nitroblue tetrazolium reduction test. Am J Med 58:685, 1975. 17. Metzke, H. & Oppermann, J.: Die Diphenylaminreaktion als Aktivitatsparameter bei der juvenilen Rheumatoid-Arthritis. Dtsch Gesundheitsw 30: 1857, 1975. 18. Nathan, D. G., Baehner, R. L. & Waever, D. K.: Failure of nitroblue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest48: 1895, 1%9. 19. Nowoslawski, A. & Brzosko, W. J.: Immunopathology of rheumatoid arthritis. The rheumatoid synovitis. Pathol Eur2: 198, 1%7. 20. Nowoslawski, A. & Brzosko, W. J.: Immunpathology of rheumatoid arthritis. 11. rheumatoid nodule (the rheumatoid granuloma). Pathol Eur 2: 302, 1%7. 21. Nydegger, U . E., Miescher, A,, Anner, R. M., Creighton, D. W., Lambert, P. H. & Miescher, P. A.: Serum and cellular factor involvement in nitroblue tetrazolium (NBT) reduction by human neutrophils. Klin Wochenschr51: 377, 1973. 22. Okuda, K., Tanokoro, I. & Sekido, M.: The NBT test in Behcet’s syndrome. New Engl J Med 290: 915, 1974. 23. Pachmann, L. M., Jayanetra, P. & Rothberg. R. M.: Rheumatoid sera and soluble complexes: Nitroblue tetrazolium dye test and hexose monophosphate shunt activation. Pediatrics 52: 823, 1973. 24. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid. J Pediatrics 78: 376, 1971. 25. Park, B. H. & Good, R. A.: NBT test stimulated. Lancet 2: 616, 1970. 26. Park, B. H., Fikrig, S. M.& Smithwick, E. M.: Infection and nitroblue tetrazolium reduction by neutrophils. A diagnostic aid. Lancet 2: 532, 1%8. 27. Phelps, P.: Polymorphonuclear leukocyte motility in
Srunil J Rheumnrulogy 6
28.
29. 30. 31.
32.
33.
34.
35.
vitro. 111. Possible release of a chemotactic substance following phagocytosis of urate crystals by polymorphonuclear leukocytes. Arthritis Rheum 12: 197, 1969. Preisig, E. & Hitzig. W. H., Nitroblue-tetrazolium test for the detection of chronic granulomatous disease. Technical modification. Eur J Clin Invest 1:409. 1971. Segal, A. W.: Nitroblue-tetrazolium test. Lancet 2: 1248, 1974. Segal, A. W. & Levi, A. J.: The mechanism of entry of dye into neutrophils in the nitroblue tetrazolium (NBT)test. Clin Sci Mol Med45:817, 1973. Wenger, M. E. & Bole, G. G.: Nitroblue tetrazolium dye reduction by peripheral leukocytes from rheumatoid arthritis and systemic lupus erythematosus patients measured by a histochemical and spectrophotometric method. J Lab Clin Med82:513, 1973. Winchester, R. J . , Agnello, E. & Kunkel, H. G.: Gammaglobulin complexes in synovial fluids of patients with rheumatoid arthritis: partial characterization and relationship to lowered complement levels. Clin Exp lmmunol6: 689, 1970. Winchester, R. J., Liturin, S. D., Koftler, D. & Kunkel, H. G.: Arthritis Rheum 14:650, 1971. Ref.: Eibl, M.: Nachweis der cellularen Immunitat gegen homologes und autologes, aggregiertes sowie gegen komplexgebundenes IgG bei primar chronischer Polyarthritis. Z Immunitaetsforsch Allerg Klin Immunol 144: 103, 1972. Windhorst, D. B., Holmes, B. & Good, R. A.: A newly defined x-linked trait in man with demonstration of the lyon effection carrier females. Lancet I: 737, 1%7. Zucker-Franklin, D.: The phagosomes in rheumatoid synovial fluid leukocytes: A light fluorescence and electron microscope study. Arthritis Rheum 9: 24, 1966.
Submitted for publication August 19, 1976
M. John Pediatric Clinic Martin-Luther-University Halle-Wittenberg
