H. W. KSlmel, T. Egri
The Nitroblue-Tetrazolium Test in Granulocytes of the Cerebrospinal Fluid - Methodological Problems Summary: It was originally our intention to study the suitability of the nitroblue-tetrazolium (NBT) test for differentiating the causes of granulocytic pleocytosis of the cerebrospinal fluid (CSF). However, several methodological problems were encountered and required extensive preliminary studies. The test was initially performed on 51 CSF samples from 37 patients using the method described by Park et al. in 1968. Although bacterial meningitis was demonstrated in 19 patients, the NBT test resulted in more than 11% NBT positive granulocytes in only four cases. No NBT positive cells were found in 24 of the samples tested. These rather poor results are not surprising if one considers that NBT is only taken up by granulocytes as macrocomplex containing heparin or fibrinogen. CSF does not contain measureabIe quantities of fibrinogen-either under normal conditions or in cases of viral or bacterial meningitis. A comparative study of the NBT test with and without the addition of heparin was performed using CSF samples from six patients with proven bacterial meningitis. Without heparin there were less than ll~/0 NBT positive granulocytes in five cases, while all samples demonstrated more than 11% positive cells after addition of heparin. Best results were achieved with 70 IU of heparin in each sample. Preparation and evaluation were facilitated by use of 0.1~/0 NBT solution and concentration of the CSF cells in a sedimentation chamber.
Zusammenfassung: Der Nitroblau-Tetrazolium-Test an Granulozyten des Liquor cerebrospinalis. Methodische Probleme. Die Brauchbarkeit des Nitroblau-Tetrazolium-Tests (NBT) zur Differenzierung yon granulozyt~iren Pleozytosen des Liquor cerebrospinalis sollte bestimmt werden. Dabei ergaben sich methodische Schwierigkeiten, die zahlreiche Voruntersuchungen notwendig machten. Zuniichst wurde der Test in der von Park et al. I968 angegegebenen Methode an 51 Liquorproben yon 37 Patienten durchgeffihrt. Obwohl bei 19 dieser Patienten eine bakterielle Meningitis nachgewiesen werden konnte, zeigte der Test nut viermal mehr als 11~/0 der Granulozyten NBT-positiv. Bei 24 Liquorproben lag die Zahl NBT-positiver Granulozyten sogar bei 00/0. Dieses vergleichbar schlechte Ergebnis verwundert nicht, wenn man berticksichtigt, dab das NBT nur als MakrokompIex mit Heparin oder Fibrinogen yon den Granulozyten aufgenommen werden kann. Liquor enthiilt jedoch weder unter Normalbedingungen noch bei viraler oder bakterieller Meningitis fagbare Fibrinogenmengen. In einer weiteren Studie wurde deshalb bei sechs Patienten mit nachgewiesener bakterieller Meningitis einmaI der Test ohne Zugabe yon Heparin, einreal unter Zugabe yon Heparin durchgefiihrt. Wiihrend bei fehlendem Heparin ffinf Ergebnisse unter 11% positiver Granulozyten lagen, zeigten nach Hinzugabe yon Heparin alle Proben z.T. welt fiber 11% NBT-positive Granulozyten. Als optimale Heparinmenge el~,ies sich 70 IU. Das Ergebnis bzw. die Auswertung des Testes wurden schlieBlich giinstig beeinflul3t, wenn die verwendete NBT-L6sung 0.1 prozentig war, und wenn zur Konzentration der Liquorzellen eine Sedimentierkammer verweudet wurde.
10
Infection 7 (1979) Nr. 1
Introduction N i t r o b l u e - t e t r a z o l i u m (NBT) dye is a colorless salt which is taken up by healthy juvenile and m a t u r e neutrophils, reduced and stored in the cytoplasm in the f o r m of blueblack formazan, as a function of the oxydase activity of the cells (1, 2). T h e p r o p o r t i o n of N B T positive granulocytes is low in healthy individuals and in patients with viral infections, but high in patients with acute bacterial disease. Park et al. (2) and other authors (3, 4, 5) have shown that the probability of bacterial infection is high w h e n 1 1 % or m o r e of the granulocytes are N B T positive. T h e m a j o r i t y of authors claim that the N B T test is inadequate as a diagnostic blood test for bacterial meningitis (6, 7, 8). It would seem that most cases of bacterial meningitis present as localized infections for purposes of the N B T test, with n o r m a l test results despite generalized symptoms. A similar p h e n o m e n o n has b e e n described in streptococcal pharyngitis (9). It is surprising that we have only a few reports to date on use of the N B T test in granulocytes of the C S F (8, 10, 11). W e h a v e attempted to evaluate the N B T test in granulocytes of the C S F as a m e t h o d for diagnosing bacterial meningitis. W e encountered a n u m b e r of m e t h o d o l o g i c a l problems, as the following report will show.
Materials and Methods The NBT test was employed in patients who presented with the clinical picture of infection of the central nervous system and with a CSF cell count of more than 150 cells per ml. In the majority of cases, CSF was collected in sterile test tubes by means of lumbar puncture, though ventricular puncture was occasionally employed. The CS'F was incubated with NBT solution, and cells were concentrated in a modified sedimentation chamber (12). In one series, cells were concentrated with a cytocentrifuge. Cell evaluation was performed under a microscope (x 1000). Granulocytes were considered NBT positive if they contained a large homogeneous formazan deposit or a large number of small formazan deposits in the cytoplasm. Ceils containing no formazan or only a few small formazan deposits were considered NBT negative. Monocytes and lymphocytes were not included in the results. One hundred granulocytes were counted and the number of NBT positive ceils recorded as a percentage. Eleven percent or more, reported by Park et al. (2) as significant for bacterial infection, were adopted initially for the CSF studies.
Received: 17 August 1978 Ass. Prof. Dr. H. W. KOlmel, Dr. T. Egri, Neurologische Abteilung des KliniktLm Charlottenburg der Freien Universidit, Spandauer Datum 130, D-1000 Berlin 19,
H. W. KOlmel, T. Egri: The Nitroblue-Tetrazoiium 'rest in Granuiocytes of the Cerebrospinal Fluid %
N B T Test According to Park The test was first performed using the method described by Park et al. (2). The authors who had employed the test on CSF granulocytes followed the same procedure (8, 10, 11). The following steps are involved in the quantitative cytological NBT test without stimulation: - Preparation of a 0.2~/0 N B T solution using N B T dye and normal saline. - Mixing of equal parts of 0.2~/o NBT solution and phosphate buffer to produce the working N B T solution. - Addition of 1 ml of working solution to 1 ml of CSF. - Incubation at 37 °C for 25 min (water bath incubator). - Cell concentration and cooling for 15 min at room temperature. Staining according to Pappenheim.
o
NBT- positive granulocytes
20 0 0
15 z
-q -1 10
QOII
8' e-
Fifty-one CSF samples from 37 patients were evaluated with the N B T test. Bacteria were identified in 19 cases. In the remaining 18 cases, bacterial meningitis was suspected despite negative bacteriological studies, or else meningitis of other etiology-viral, foreign-body, etc.-was demonstrated at some point. Thirty-two CSF samples were tested in the 19 cases of proven bacterial meningitis, and only four of the samples produced values greater than 11~/~ N B T positive cells. Fourteen of 32 sampIes showed no positive granulocytes at all (Figure 1). On the basis of these findings, the N B T test would appear to be unsuited for differentiation of inflammatory disease of the central nervous system.
N B T Test according to Park with Heparin Inspired by the work of Segal and Levi (13) and Czarnetzki et aI. (14), and making use of the fact that the CSF contains no fibrinogen (15), we performed another series of studies in which heparin was added to the N B T working solution. The optimum heparin concentration was determined in a preliminary study, with the result that 70 I U of heparin per 1 ml
O0 fOOl llI OI OOlB OSO0
-
5
00
-t -t -t
0J
OIO0000000000011IBDDDDDDD
Figure 1: Results o[ 51 CSF samples #rom 37 patients using the N B T test described by Park et al. (1968). Q) Proven bacterial meningitis. No blood in the CSF. ® Proven bacterial meningitis. Blood in the CSF. E~] Meningitis of diverse etiology (virus, tuberculosis, foreign body) or suspicion of bacterial meningitis (without demonstration of the organism). No blood in the CSF. [] Meningitis of diverse etiology or suspicion of bacterial meningitis. Blood in the CSF. of CSF appeared most useful. Higher heparin concentrations resulted in extracellular heparin-NBT complexes which produced a violet precipitate and severely disturbed evaluation of the slide preparations.
Table 1: Results of the N B T test in CSF granulocytes with and without addition of heparin.
Patient
Age
Diagnosis
Duration Number of illness of cells/ccm
Protein mg/100 ml
Glucose mg/100 ml
NBT pos. granulocytes (%) without heparin
N B T pos. granulocytes (%) with 70 I U heparin
1.
M.K.
17
bacterial meningitis; pneumococcus demonstrated in CSF
2 d
27000
580
0
10
27
2.
S.O.
81
bacterial meningitis; pneumococcus demonstrated in CSF
1 d
12800
808
15
2
23
3.
St. H.
51
bacterial meningitis; pneumococcus demonstrated in CSF
2 d
3200
450
10
5
23
4.
S.H.
52
meningitis following neurosurgery; Staphylococcus pyogenes
30 d
3120
308
93
7
40
5.
W.O.
19
subdural empyema; Enterobacter aerogenes isolated in CSF culture
44 d
760
335
10
i2
39
6.
B.S.
64
bacterial meningitis; pneumococcus demonstrated in CSF
1 d
4800
640
0
0
20
Infection 7 (1979) Nr. 1
11
H. W. K61mel, T. Egri: The Nitroblue-Tetrazolium Test in Granulocytes of the Cerebrospinal Fluid Table 2: Percentage of N B T positive granulocytes with different N B T concentrations. Addition of 70 IU heparin to CSF/NBT.
Patient
Age
Diagnosis
Duration of illness
Number of cells/ccm
Protein mg/100 mI
NBT pos. granulocytes (°/a) with 0.1°/0 NBT solution
NBT pos. granulocytes (°/0) with 0.20/0 NBT solution
7.
W.O.
19
subdural empyema; Enterobacter aerogenes isolated in CSF culture
51 d
890
350
32
19
8.
K.K.
67
foreign body meningitis
5 d
100
266
12
3
9.
K.H.
64
bacterial meningitis; Enterobaeter aerogenes isolated in CSF culture
6 d
85
220
21
15
10.
B.O.
70
bacterial meningitis; pneumococcus demonstrated in CSF
3 d
3300
450
16
22
11.
M.U.
28
bacterial meningitis; pneumococcus demonstrated in CS'F
9 d
183
57
26
18
12.
T.O.
15
foreign body meningitis
3 d
50
140
7
6
CSF from six patients with proven bacterial meningitis was studied, once using Park's method without heparin and once with the addition of 70 I U of heparin per 1 ml of CSF. Table 1 shows that more than 11~/0 of the granutocytes appeared to be NBT positive in all six cases after addition of heparin. Without heparin, five samples showed less than 11% NBT positive cells, while the sixth case resulted in I20/0 of granuIocytes containing formazan. Alteration of the N B T Concentration Since some investigators have found that a decrease in the NBT concentration from 0.2% to 0.1% improves the percentage of formazan positive cells, we performed a series in order to study the influence of NBT concentration on granulocytes of the CSIL As Table 2 shows, it is quite likely that a reduction of the NBT concentration to 0.1°/0 improves results with CSF granulocytes. Ceil Concentration with the Cytoeentrifuge and with the Sedimentation Method According to studies by Gordon et al. (8), concentration of blood cells with the cytocentrifuge allows especially good results with the NBT test. In our series, CSF samples from patients with clinical signs of bacterial meningitis were prepared using a modification of Park's method (70 IU heparin/ ml CSF; 0.1% NBT concentration) and subsequently concentrated with the cytocentrifuge. Samples from the same group of patients were concentrated in the sedimentation chamber according to Sayk (2). Cytologic evaluation was performed under the microscope in the usual manner. In six cases the number of NBT positive granulocytes was higher after concentration in the sedimentation chamber, and in three cases the number of N B T positive granulocytes was greater after concentration in the cytocentrifuge. The results were identical in one case. In an additional five cases cells were studied only after concentration in the sedimentation chamber, since cells concentrated with the cytocentrifuge were too severely altered for evaluation. On the basis of these results, cell concentration with the sedimentation chamber is superior to that with the cytocentrifuge.
19
Infection 7 (1979) Nr. 1
Discussion Esposito and Detlala (1.0) were the first to report on the N B T test with C S F granuloeytes. As other authors have shown (6, 7, 8), the test is useful in differentiation of various forms of meningitis. T h e seven cases of bacterial meningitis studied by these authors clearly demonstrated positive results (18--100o/0 N B T positive cells). Gordon et al. (8) m a d e a brief report on similar findings, though they did not provide specific figures. Zwiebel and Schwarzman published the first extensive report on the N B T test with neutrophils of the C S F (11). I n their study, results were positive in all ten cases of p r o v e n bacterial meningitis. "['he N B T test was p e r f o r m e d before initiation of antibiotic therapy or within the first 14 days of antibiotic therapy. T w o patients suffered cerebral hemorrhage, and the N B T test was negative in these cases. All authors used Park's method. W e e m p l o y e d Park's m e t h o d in a large series, but the results do not coincide with those of the other authors. T h e percentage of N B T positive cells exceeded 110/0 in only f o u r cases, and in 14 cases no granulocytes appeared N B T positive. Since the test was developed for blood granulocytes, one must assume that the p o o r results with granulocytes of the C S F derive f r o m the absence of a necessary factor in the CSF. It had been assumed that activation of the granulocytes was coupled with increased permeability, such that N B T dye could simply penetrate the cell m e m brane (16, 17). Park and Good (18) postulated that complement was necessary f o r intracellular reduction of N B T dye to f o r m a z a n , but Segal and Levi (13) showed that c o m p l e m e n t was necessary for transport of N B T across the cell m e m b r a n e . Czarnetzki et al. found that granulo-
H, W, KiSlmel, T. Egri: The Nitroblue-Tetrazolium Test in Granulocytes of the CerebrospinaI Fluid cytes take up N B T dye only in macrocomplex form with heparin or fibrinogen (14). One might conclude that the failure of our N B T test in granulocytes of the CSF results from the absence of fibrinogen. In all four cases in which we found more than t10/0 N B T positive cells (Figure 1), there was blood and therefore a certain amount of fibrinogen in the CSF. The positive results might be explained by that fact. All six cases of proven bacterial meningitis showed positive results after the addition of heparin (70 I U / m l CSF). Without heparin, the results were positive in only one case. Therefore we believe that the results of the test, as performed by the aforementioned authors on granulocytes of the CSF, are not reproducible, since neither heparin nor fibrinogen was added to the CSF. The absence of further reports on the use of this test on CSF granulocytes may be due to the fact that the test does not provide satisfactory results when performed according to the method described by Park. Several researchers found that reduction of the N B T concentration from 0.2% to 0.10/0 increased the percentage of N B T positive cells (3, 19). We performed a comparative study on the two concentrations in the N B T test and found a higher percentage of N B T positive cells in five of six cases with 0A0/0 NBT. There is no complete explanation for this difference.
Further studies will determine the value of the N B T test in granulocytes of the CSF for differentiation of meningitis.
Literature 1. Baehner, R., Nathan, D. G.: Leukocyte oxidase: Defective activity in chronic granulomatous disease. Science 155 (1967) 835-836. 2. Park, B. H., Fikrig, S. M., Smithwick, E. M.: Infection and NBT reduction by neutrophils, a diagnostic aid. Lancet 1 (1968) 532-535. 3. Humbert, J. R., Melvin, M., Hathaway, E., Thoren, Ch.: The histochemical NBT reduction test in the differential diagnosis of acute infections. Pediatrics 48 (1971) 259-267. 4. Matula, G., Peterson, P. Y.: Spontaneous in vitro reduction of NBT by neutrophils of adult patients with bacterial infection. N. Engl. J. Med. 285 (1971) 311-317. 5. Vaucher, A., Wyss, M., Thevoz, F., Kniip]el, M., Miescher, P. A.: La r6duction du NBT par les granulocytes dans divers etats cliniques. Schweiz. med. Wschr. 100 (1970) 2248-2251. 6. Feigin, R. D., Shackelford, P. G., Choi, S. C., Flake, Franklin, Eisenberg: NBT dye test as an aid in the differentim diagnosis of febrile disorders. J. Pediatr. 78 (1971) 230237. 7. Giidl, J.: Der Nitroblau-Tetrazolium-Test in der Diagnostik der Meningitis purulenta. Infection 4 (1976) 212-214.
N B T tends to crystallize with increasing concentration. Segall and Levi (13) demonstrated that crystalline N B T is not taken up by granulocytes in significant amounts, so that little or no reduction of the substance takes place. Finally we compared the cytological results in the N B T test after concentration with the cytocentrifuge and the sedimentation chamber. It would seem that cells of the CSF, which are quite fragile (12), respond to mechanical forces with morphological changes, especially after addition of the cytotoxic N B T dye and incubation at 37 °C. The cytocentrifuge frequently causes such extensive alteration that many cell preparations can no longer be studied. In contrast to the results obtained by Gordon et al. in concentrating blood granulocytes (8), we found the sedimentation method after Sayk better suited for concentrating celIs of the CSF. We propose the following modification of the N B T test for use with granulocytes of the CSF:
8. Gordon, A. M., Rowan, R. M., Brown, T., Carson, H. G.: Routine application of the NBT-test in the clinical laboratory. J. Clin. Pathol. 26 (1973) 52-56. 9. Shapera, R. M., Matsen, J.: NBT dye reduction by neutrophils from patients with streptococcal pharyngitis. Pediatrics 51 (1973) 284-287. 10. Esposito, R., De Lalla, F.: NBT-test in bacterial meningitis. Lancet 1 (1972) 747-748. 11. Zwibel; H. L., Schwartzman, R. G.: Evaluation of the NBT test as applied to polymorphnuclear leukocytes in the cerebrospinal fluid. Neurology 24 (1974) 995-997.
-- Preparation of a 0.10/0 N B T solution from N B T dye and normal saline.
15. Brueton, M. J., Breese, G. R., Stuart, J.: Fibrin-fibrinogen degradation products in cerebrospinal fluid. J. Clin. Path. 65 (1976) 341-344. 16. Nathan, D. G., Baehner, R. L., Weaver, D. K.: Failure of NBT reduction in the phagocytic vacuoles of leukocytes in CGD. J. Clin. Invest. 48 (1969) 1895-1904.
Preparation of a working-NBT solution from equal parts of 0.1% N B T solution and phosphate buffer.
-
- Addition of 1 ml of CSF to 1 ml working solution with 70 I U heparin. Incubation at 37 °C for 25 min in a water bath incubator.
-
- Cell concentration in a sedimentation chamber for 15 min at room temperature. -
Air drying of the slide followed by Pappenheim stain.
12. KOhnet, H. W.: Atlas of cerebrospinal fluid cells. Springer, Berlin/Heidelberg/New York 1977. 13. SegaI, A. W., Levi, A.: The mechanism of the entry of dye into neutrophils in the NBT test. Clin. Sc. Moi. Med. 45 (1973) 817-825. 14. Czarnetzki, B. M., Cowan, D. H., Belcher, R.: The effects of polyanious on NBT reduction hexose monophosphate shunt activitiy and ultrastructure of polymorphnuclear leukocytes. Am. J. Clin. Path. 64 (1975) 34-40.
17. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid. J. Pediatr. 78 (1971) 376378. 18. Park, B. H., Good, R. A.: NBT test stimulated. Lancet 2 (1970) 616. 19. Segal, A. W., Trustey, S. F., Levi, A. J.: Re-Evaluation of the NBT test. Lancet 2 (1973) 879-883.
Infection 7 (1979) Nr. 1
13
The Nitroblue-Tetrazolium Test in Granulocytes of the Cerebrospinal Fluid - Methodological Problems Summary: It was originally our intention to study the suitability of the nitroblue-tetrazolium (NBT) test for differentiating the causes of granulocytic pleocytosis of the cerebrospinal fluid (CSF). However, several methodological problems were encountered and required extensive preliminary studies. The test was initially performed on 51 CSF samples from 37 patients using the method described by Park et al. in 1968. Although bacterial meningitis was demonstrated in 19 patients, the NBT test resulted in more than 11% NBT positive granulocytes in only four cases. No NBT positive cells were found in 24 of the samples tested. These rather poor results are not surprising if one considers that NBT is only taken up by granulocytes as macrocomplex containing heparin or fibrinogen. CSF does not contain measureabIe quantities of fibrinogen-either under normal conditions or in cases of viral or bacterial meningitis. A comparative study of the NBT test with and without the addition of heparin was performed using CSF samples from six patients with proven bacterial meningitis. Without heparin there were less than ll~/0 NBT positive granulocytes in five cases, while all samples demonstrated more than 11% positive cells after addition of heparin. Best results were achieved with 70 IU of heparin in each sample. Preparation and evaluation were facilitated by use of 0.1~/0 NBT solution and concentration of the CSF cells in a sedimentation chamber.
Zusammenfassung: Der Nitroblau-Tetrazolium-Test an Granulozyten des Liquor cerebrospinalis. Methodische Probleme. Die Brauchbarkeit des Nitroblau-Tetrazolium-Tests (NBT) zur Differenzierung yon granulozyt~iren Pleozytosen des Liquor cerebrospinalis sollte bestimmt werden. Dabei ergaben sich methodische Schwierigkeiten, die zahlreiche Voruntersuchungen notwendig machten. Zuniichst wurde der Test in der von Park et al. I968 angegegebenen Methode an 51 Liquorproben yon 37 Patienten durchgeffihrt. Obwohl bei 19 dieser Patienten eine bakterielle Meningitis nachgewiesen werden konnte, zeigte der Test nut viermal mehr als 11~/0 der Granulozyten NBT-positiv. Bei 24 Liquorproben lag die Zahl NBT-positiver Granulozyten sogar bei 00/0. Dieses vergleichbar schlechte Ergebnis verwundert nicht, wenn man berticksichtigt, dab das NBT nur als MakrokompIex mit Heparin oder Fibrinogen yon den Granulozyten aufgenommen werden kann. Liquor enthiilt jedoch weder unter Normalbedingungen noch bei viraler oder bakterieller Meningitis fagbare Fibrinogenmengen. In einer weiteren Studie wurde deshalb bei sechs Patienten mit nachgewiesener bakterieller Meningitis einmaI der Test ohne Zugabe yon Heparin, einreal unter Zugabe yon Heparin durchgefiihrt. Wiihrend bei fehlendem Heparin ffinf Ergebnisse unter 11% positiver Granulozyten lagen, zeigten nach Hinzugabe yon Heparin alle Proben z.T. welt fiber 11% NBT-positive Granulozyten. Als optimale Heparinmenge el~,ies sich 70 IU. Das Ergebnis bzw. die Auswertung des Testes wurden schlieBlich giinstig beeinflul3t, wenn die verwendete NBT-L6sung 0.1 prozentig war, und wenn zur Konzentration der Liquorzellen eine Sedimentierkammer verweudet wurde.
10
Infection 7 (1979) Nr. 1
Introduction N i t r o b l u e - t e t r a z o l i u m (NBT) dye is a colorless salt which is taken up by healthy juvenile and m a t u r e neutrophils, reduced and stored in the cytoplasm in the f o r m of blueblack formazan, as a function of the oxydase activity of the cells (1, 2). T h e p r o p o r t i o n of N B T positive granulocytes is low in healthy individuals and in patients with viral infections, but high in patients with acute bacterial disease. Park et al. (2) and other authors (3, 4, 5) have shown that the probability of bacterial infection is high w h e n 1 1 % or m o r e of the granulocytes are N B T positive. T h e m a j o r i t y of authors claim that the N B T test is inadequate as a diagnostic blood test for bacterial meningitis (6, 7, 8). It would seem that most cases of bacterial meningitis present as localized infections for purposes of the N B T test, with n o r m a l test results despite generalized symptoms. A similar p h e n o m e n o n has b e e n described in streptococcal pharyngitis (9). It is surprising that we have only a few reports to date on use of the N B T test in granulocytes of the C S F (8, 10, 11). W e h a v e attempted to evaluate the N B T test in granulocytes of the C S F as a m e t h o d for diagnosing bacterial meningitis. W e encountered a n u m b e r of m e t h o d o l o g i c a l problems, as the following report will show.
Materials and Methods The NBT test was employed in patients who presented with the clinical picture of infection of the central nervous system and with a CSF cell count of more than 150 cells per ml. In the majority of cases, CSF was collected in sterile test tubes by means of lumbar puncture, though ventricular puncture was occasionally employed. The CS'F was incubated with NBT solution, and cells were concentrated in a modified sedimentation chamber (12). In one series, cells were concentrated with a cytocentrifuge. Cell evaluation was performed under a microscope (x 1000). Granulocytes were considered NBT positive if they contained a large homogeneous formazan deposit or a large number of small formazan deposits in the cytoplasm. Ceils containing no formazan or only a few small formazan deposits were considered NBT negative. Monocytes and lymphocytes were not included in the results. One hundred granulocytes were counted and the number of NBT positive ceils recorded as a percentage. Eleven percent or more, reported by Park et al. (2) as significant for bacterial infection, were adopted initially for the CSF studies.
Received: 17 August 1978 Ass. Prof. Dr. H. W. KOlmel, Dr. T. Egri, Neurologische Abteilung des KliniktLm Charlottenburg der Freien Universidit, Spandauer Datum 130, D-1000 Berlin 19,
H. W. KOlmel, T. Egri: The Nitroblue-Tetrazoiium 'rest in Granuiocytes of the Cerebrospinal Fluid %
N B T Test According to Park The test was first performed using the method described by Park et al. (2). The authors who had employed the test on CSF granulocytes followed the same procedure (8, 10, 11). The following steps are involved in the quantitative cytological NBT test without stimulation: - Preparation of a 0.2~/0 N B T solution using N B T dye and normal saline. - Mixing of equal parts of 0.2~/o NBT solution and phosphate buffer to produce the working N B T solution. - Addition of 1 ml of working solution to 1 ml of CSF. - Incubation at 37 °C for 25 min (water bath incubator). - Cell concentration and cooling for 15 min at room temperature. Staining according to Pappenheim.
o
NBT- positive granulocytes
20 0 0
15 z
-q -1 10
QOII
8' e-
Fifty-one CSF samples from 37 patients were evaluated with the N B T test. Bacteria were identified in 19 cases. In the remaining 18 cases, bacterial meningitis was suspected despite negative bacteriological studies, or else meningitis of other etiology-viral, foreign-body, etc.-was demonstrated at some point. Thirty-two CSF samples were tested in the 19 cases of proven bacterial meningitis, and only four of the samples produced values greater than 11~/~ N B T positive cells. Fourteen of 32 sampIes showed no positive granulocytes at all (Figure 1). On the basis of these findings, the N B T test would appear to be unsuited for differentiation of inflammatory disease of the central nervous system.
N B T Test according to Park with Heparin Inspired by the work of Segal and Levi (13) and Czarnetzki et aI. (14), and making use of the fact that the CSF contains no fibrinogen (15), we performed another series of studies in which heparin was added to the N B T working solution. The optimum heparin concentration was determined in a preliminary study, with the result that 70 I U of heparin per 1 ml
O0 fOOl llI OI OOlB OSO0
-
5
00
-t -t -t
0J
OIO0000000000011IBDDDDDDD
Figure 1: Results o[ 51 CSF samples #rom 37 patients using the N B T test described by Park et al. (1968). Q) Proven bacterial meningitis. No blood in the CSF. ® Proven bacterial meningitis. Blood in the CSF. E~] Meningitis of diverse etiology (virus, tuberculosis, foreign body) or suspicion of bacterial meningitis (without demonstration of the organism). No blood in the CSF. [] Meningitis of diverse etiology or suspicion of bacterial meningitis. Blood in the CSF. of CSF appeared most useful. Higher heparin concentrations resulted in extracellular heparin-NBT complexes which produced a violet precipitate and severely disturbed evaluation of the slide preparations.
Table 1: Results of the N B T test in CSF granulocytes with and without addition of heparin.
Patient
Age
Diagnosis
Duration Number of illness of cells/ccm
Protein mg/100 ml
Glucose mg/100 ml
NBT pos. granulocytes (%) without heparin
N B T pos. granulocytes (%) with 70 I U heparin
1.
M.K.
17
bacterial meningitis; pneumococcus demonstrated in CSF
2 d
27000
580
0
10
27
2.
S.O.
81
bacterial meningitis; pneumococcus demonstrated in CSF
1 d
12800
808
15
2
23
3.
St. H.
51
bacterial meningitis; pneumococcus demonstrated in CSF
2 d
3200
450
10
5
23
4.
S.H.
52
meningitis following neurosurgery; Staphylococcus pyogenes
30 d
3120
308
93
7
40
5.
W.O.
19
subdural empyema; Enterobacter aerogenes isolated in CSF culture
44 d
760
335
10
i2
39
6.
B.S.
64
bacterial meningitis; pneumococcus demonstrated in CSF
1 d
4800
640
0
0
20
Infection 7 (1979) Nr. 1
11
H. W. K61mel, T. Egri: The Nitroblue-Tetrazolium Test in Granulocytes of the Cerebrospinal Fluid Table 2: Percentage of N B T positive granulocytes with different N B T concentrations. Addition of 70 IU heparin to CSF/NBT.
Patient
Age
Diagnosis
Duration of illness
Number of cells/ccm
Protein mg/100 mI
NBT pos. granulocytes (°/a) with 0.1°/0 NBT solution
NBT pos. granulocytes (°/0) with 0.20/0 NBT solution
7.
W.O.
19
subdural empyema; Enterobacter aerogenes isolated in CSF culture
51 d
890
350
32
19
8.
K.K.
67
foreign body meningitis
5 d
100
266
12
3
9.
K.H.
64
bacterial meningitis; Enterobaeter aerogenes isolated in CSF culture
6 d
85
220
21
15
10.
B.O.
70
bacterial meningitis; pneumococcus demonstrated in CSF
3 d
3300
450
16
22
11.
M.U.
28
bacterial meningitis; pneumococcus demonstrated in CS'F
9 d
183
57
26
18
12.
T.O.
15
foreign body meningitis
3 d
50
140
7
6
CSF from six patients with proven bacterial meningitis was studied, once using Park's method without heparin and once with the addition of 70 I U of heparin per 1 ml of CSF. Table 1 shows that more than 11~/0 of the granutocytes appeared to be NBT positive in all six cases after addition of heparin. Without heparin, five samples showed less than 11% NBT positive cells, while the sixth case resulted in I20/0 of granuIocytes containing formazan. Alteration of the N B T Concentration Since some investigators have found that a decrease in the NBT concentration from 0.2% to 0.1% improves the percentage of formazan positive cells, we performed a series in order to study the influence of NBT concentration on granulocytes of the CSIL As Table 2 shows, it is quite likely that a reduction of the NBT concentration to 0.1°/0 improves results with CSF granulocytes. Ceil Concentration with the Cytoeentrifuge and with the Sedimentation Method According to studies by Gordon et al. (8), concentration of blood cells with the cytocentrifuge allows especially good results with the NBT test. In our series, CSF samples from patients with clinical signs of bacterial meningitis were prepared using a modification of Park's method (70 IU heparin/ ml CSF; 0.1% NBT concentration) and subsequently concentrated with the cytocentrifuge. Samples from the same group of patients were concentrated in the sedimentation chamber according to Sayk (2). Cytologic evaluation was performed under the microscope in the usual manner. In six cases the number of NBT positive granulocytes was higher after concentration in the sedimentation chamber, and in three cases the number of N B T positive granulocytes was greater after concentration in the cytocentrifuge. The results were identical in one case. In an additional five cases cells were studied only after concentration in the sedimentation chamber, since cells concentrated with the cytocentrifuge were too severely altered for evaluation. On the basis of these results, cell concentration with the sedimentation chamber is superior to that with the cytocentrifuge.
19
Infection 7 (1979) Nr. 1
Discussion Esposito and Detlala (1.0) were the first to report on the N B T test with C S F granuloeytes. As other authors have shown (6, 7, 8), the test is useful in differentiation of various forms of meningitis. T h e seven cases of bacterial meningitis studied by these authors clearly demonstrated positive results (18--100o/0 N B T positive cells). Gordon et al. (8) m a d e a brief report on similar findings, though they did not provide specific figures. Zwiebel and Schwarzman published the first extensive report on the N B T test with neutrophils of the C S F (11). I n their study, results were positive in all ten cases of p r o v e n bacterial meningitis. "['he N B T test was p e r f o r m e d before initiation of antibiotic therapy or within the first 14 days of antibiotic therapy. T w o patients suffered cerebral hemorrhage, and the N B T test was negative in these cases. All authors used Park's method. W e e m p l o y e d Park's m e t h o d in a large series, but the results do not coincide with those of the other authors. T h e percentage of N B T positive cells exceeded 110/0 in only f o u r cases, and in 14 cases no granulocytes appeared N B T positive. Since the test was developed for blood granulocytes, one must assume that the p o o r results with granulocytes of the C S F derive f r o m the absence of a necessary factor in the CSF. It had been assumed that activation of the granulocytes was coupled with increased permeability, such that N B T dye could simply penetrate the cell m e m brane (16, 17). Park and Good (18) postulated that complement was necessary f o r intracellular reduction of N B T dye to f o r m a z a n , but Segal and Levi (13) showed that c o m p l e m e n t was necessary for transport of N B T across the cell m e m b r a n e . Czarnetzki et al. found that granulo-
H, W, KiSlmel, T. Egri: The Nitroblue-Tetrazolium Test in Granulocytes of the CerebrospinaI Fluid cytes take up N B T dye only in macrocomplex form with heparin or fibrinogen (14). One might conclude that the failure of our N B T test in granulocytes of the CSF results from the absence of fibrinogen. In all four cases in which we found more than t10/0 N B T positive cells (Figure 1), there was blood and therefore a certain amount of fibrinogen in the CSF. The positive results might be explained by that fact. All six cases of proven bacterial meningitis showed positive results after the addition of heparin (70 I U / m l CSF). Without heparin, the results were positive in only one case. Therefore we believe that the results of the test, as performed by the aforementioned authors on granulocytes of the CSF, are not reproducible, since neither heparin nor fibrinogen was added to the CSF. The absence of further reports on the use of this test on CSF granulocytes may be due to the fact that the test does not provide satisfactory results when performed according to the method described by Park. Several researchers found that reduction of the N B T concentration from 0.2% to 0.10/0 increased the percentage of N B T positive cells (3, 19). We performed a comparative study on the two concentrations in the N B T test and found a higher percentage of N B T positive cells in five of six cases with 0A0/0 NBT. There is no complete explanation for this difference.
Further studies will determine the value of the N B T test in granulocytes of the CSF for differentiation of meningitis.
Literature 1. Baehner, R., Nathan, D. G.: Leukocyte oxidase: Defective activity in chronic granulomatous disease. Science 155 (1967) 835-836. 2. Park, B. H., Fikrig, S. M., Smithwick, E. M.: Infection and NBT reduction by neutrophils, a diagnostic aid. Lancet 1 (1968) 532-535. 3. Humbert, J. R., Melvin, M., Hathaway, E., Thoren, Ch.: The histochemical NBT reduction test in the differential diagnosis of acute infections. Pediatrics 48 (1971) 259-267. 4. Matula, G., Peterson, P. Y.: Spontaneous in vitro reduction of NBT by neutrophils of adult patients with bacterial infection. N. Engl. J. Med. 285 (1971) 311-317. 5. Vaucher, A., Wyss, M., Thevoz, F., Kniip]el, M., Miescher, P. A.: La r6duction du NBT par les granulocytes dans divers etats cliniques. Schweiz. med. Wschr. 100 (1970) 2248-2251. 6. Feigin, R. D., Shackelford, P. G., Choi, S. C., Flake, Franklin, Eisenberg: NBT dye test as an aid in the differentim diagnosis of febrile disorders. J. Pediatr. 78 (1971) 230237. 7. Giidl, J.: Der Nitroblau-Tetrazolium-Test in der Diagnostik der Meningitis purulenta. Infection 4 (1976) 212-214.
N B T tends to crystallize with increasing concentration. Segall and Levi (13) demonstrated that crystalline N B T is not taken up by granulocytes in significant amounts, so that little or no reduction of the substance takes place. Finally we compared the cytological results in the N B T test after concentration with the cytocentrifuge and the sedimentation chamber. It would seem that cells of the CSF, which are quite fragile (12), respond to mechanical forces with morphological changes, especially after addition of the cytotoxic N B T dye and incubation at 37 °C. The cytocentrifuge frequently causes such extensive alteration that many cell preparations can no longer be studied. In contrast to the results obtained by Gordon et al. in concentrating blood granulocytes (8), we found the sedimentation method after Sayk better suited for concentrating celIs of the CSF. We propose the following modification of the N B T test for use with granulocytes of the CSF:
8. Gordon, A. M., Rowan, R. M., Brown, T., Carson, H. G.: Routine application of the NBT-test in the clinical laboratory. J. Clin. Pathol. 26 (1973) 52-56. 9. Shapera, R. M., Matsen, J.: NBT dye reduction by neutrophils from patients with streptococcal pharyngitis. Pediatrics 51 (1973) 284-287. 10. Esposito, R., De Lalla, F.: NBT-test in bacterial meningitis. Lancet 1 (1972) 747-748. 11. Zwibel; H. L., Schwartzman, R. G.: Evaluation of the NBT test as applied to polymorphnuclear leukocytes in the cerebrospinal fluid. Neurology 24 (1974) 995-997.
-- Preparation of a 0.10/0 N B T solution from N B T dye and normal saline.
15. Brueton, M. J., Breese, G. R., Stuart, J.: Fibrin-fibrinogen degradation products in cerebrospinal fluid. J. Clin. Path. 65 (1976) 341-344. 16. Nathan, D. G., Baehner, R. L., Weaver, D. K.: Failure of NBT reduction in the phagocytic vacuoles of leukocytes in CGD. J. Clin. Invest. 48 (1969) 1895-1904.
Preparation of a working-NBT solution from equal parts of 0.1% N B T solution and phosphate buffer.
-
- Addition of 1 ml of CSF to 1 ml working solution with 70 I U heparin. Incubation at 37 °C for 25 min in a water bath incubator.
-
- Cell concentration in a sedimentation chamber for 15 min at room temperature. -
Air drying of the slide followed by Pappenheim stain.
12. KOhnet, H. W.: Atlas of cerebrospinal fluid cells. Springer, Berlin/Heidelberg/New York 1977. 13. SegaI, A. W., Levi, A.: The mechanism of the entry of dye into neutrophils in the NBT test. Clin. Sc. Moi. Med. 45 (1973) 817-825. 14. Czarnetzki, B. M., Cowan, D. H., Belcher, R.: The effects of polyanious on NBT reduction hexose monophosphate shunt activitiy and ultrastructure of polymorphnuclear leukocytes. Am. J. Clin. Path. 64 (1975) 34-40.
17. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid. J. Pediatr. 78 (1971) 376378. 18. Park, B. H., Good, R. A.: NBT test stimulated. Lancet 2 (1970) 616. 19. Segal, A. W., Trustey, S. F., Levi, A. J.: Re-Evaluation of the NBT test. Lancet 2 (1973) 879-883.
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