Effectiveness of the Nitroblue Tetrazolium Test in Demonstrating Reduced Bactericidal Activity of Polymorphonuclear Neutrophils in Schistosomal Hepatosplenomegaly and Ascites M. BAGNEID, M.S., K. KAMEL, M.D., AND A. SHAKER, M.D. Research Hematology Department, U. S. Naval Medical Research unit No. 3, and Department of Medicine, Ain-Shams University Medical School, Cairo, Egypt

Bagneid, M., Kamel, K., and Shaker, A.: Effectiveness of the nitroblue tetrazolium test in demonstrating reduced bactericidal activity of polymorphonuclear neutrophils in schistosomal hepatosplenomegaly and ascites. Am J Clin Pathol 63: 921-926, 1975. T h e nitroblue tetrazolium (NBT) test was carried out on blood from ten normal individuals and 20 schistosomal patients, ten of whom had hepatosplenomegaly and ten hepatosplenomegaly and ascites. T h e test revealed defective neutrophils in patients who had hepatosplenomegaly accompanied by ascites. T h e phagocytic and bactericidal effects of the neutrophils on Staphylococcus aureus were directly measured with cells from five of each of the two groups of patients; results substantiated those obtained by the NBT screening test. T h e N B T screening test thus proved useful in detecting acquired defective function of neutrophils in a helminthic parasitic disease that is accompanied by frequent bacterial infections. (Key words: Nitroblue tetrazolium; Bactericidal activity; Polymorphonuclear neutrophils; Schistosomiasis; Hepatosplenomegaly; Ascites.)

Received August 22, 1974; received revised manuscript November 11, 1974; accepted for publication November I I , 1974. Work done at the U. S. Naval Medical Research Unit No. 3, Cairo, Egypt. Supported in part by the Bureau of Medicine and Surgery, U. S. Navy, Project Number MR 011.01 01-3007, and by the Office of Naval Research under Contract No. N000I4-70-C-0192 (NR 108-885). Presented in part at the XlVth International Congress of Hematology in Sao Paolo, Brazil, 1972. Address reprint requests to Research Publication Section, NAMRU-3, F.P.O., New York, N.Y. 09527. Dr. Kamel's present address is: Military Hospital, P.O. Box 309, Abu Dhabi, United Arab Emirates. T h e opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department, the Naval Service at large, or the Egyptian Ministry of Health. 921

THE

NITROBLUE TETRAZOLIUM

(NBT)

reduction test was introduced in 1966 for the study of polymorphonuclear leukocytes (PMN) from children with chronic granulomatous disease who die early in life from repeated infections. 1 T h e test found wide application: (1) in distinguishing bacterial infections from nonbacterial diseases accompanied by leukocytosis and fever15; (2) in detecting PMN malfunction in patients with a high risk or susceptibility to bacterial infections, e.g., those receiving steroids, 4 i m m u n o s u p p r e s s e d r e n a l t r a n s p l a n t patients, 2 4 various hematologic disorders, 5 protein-calorie

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ABSTRACT

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AJ.C.P.—Vol. 63

Table 1. Laboratory Test Results, 25 Patients with Schistosomal Hepatosplenomegaly (Mean ± 1 S.D.) Fifteen Patients with Hepatosplenomegaly and Ascites

Hemoglobin (Gm. per 100 ml.)

10.21 ± 2 . 8 1

9.82 ± 1.05

Hematocrit (%)

33.30 ± 7.69

31.67 ± 3.29

Total leukocyte count (per cu. mm.;

8,550 ± 2,794.94

4,403 ± 696.54

Neutrophils (%)

60.70 ± 9 . 1 3

57.20 ± 10.94

Lymphocytes (%)

30.10 ± 6 . 5 1

32.00 ± 8 . 2 1

Eosinophils (%)

7.60 ± 6.22

5.73 ± 3.03

Basophils (%)

1.40 ± 1.43

0.60 ± 0.74

Monocytes (%)

0.20 ± 0.42

4.47 ± 3.07

Total serum proteins (Gm. per 100 ml.) (Normal = 6 . 5 8.0 Gm. per ml.)

7.65 ± 1.30

7.55 ± 1.01

Serum albumin (Gm. per 100 ml.) (Normal = 3.8-4.9 Gm. per 100 ml.)

3.45 ± 1.07

2.62 ± 0.69

Serum globulins (Gm. per 100 ml.) (Normal = 2.1-2.6 Gm. per 100 ml.)

4.20 ± 1.01

4.93 ± 0.63

Serum bilirubin (mg. per 100 ml.) (Normal = 0.2-0.8 mg per 100 ml.)

0.58 ± 0.28

1.44 ± 0.73

12.60 ± 7.13

18.65 ± 3.89

6.63 ± 1.94

12.45 ± 4.13

SGPT (l.U. per 1.) (Normal = 15 I.U. per 1.) Alkaline phosphatase (K.-A. units per 100 ml.) (Normal = 4 - 1 2 K.-A. units per 100 ml.)

malnutrition, 2 2 and diabetes 16 ; (3) in the determination of responses to antimicrobial therapy. 13 It was further suggested that the NBT test might prove useful in Table 2. N B T Test Results with Schistosomal Hepatosplenomegaly with and without Ascites and for Normal Controls Formazan Cells (%) Range

Mean ± S.D.

Control subjects (n = 10)

55-71

60.30 ± 5.06

Patients with hepatosplenomegaly (n = 10)

42-78

63.40 ± 12.56

Patients with hepatosplenomegaly and ascites (n = 10)

19-44

28.80 ± 7.98

differentiating pneumonia from pulmonary infarctions. 9 In the majority of reports, the NBT results have not been corroborated with quantitative evaluations of bactericidal activity of PMN. T h e role of the N B T test in diagnosing bacterial infections 23 and in the routine screening of hospital populations for this purpose 3 has recently been debated. 3,23 This is an investigation into the functional integrity of PMN in human schistosomiasis, a parasitic condition complicated in its late stages by frequent infections. 21 Results of defective N B T screening test in this study have been substantiated by actual bactericidal assessment in order to evaluate the accuracy of the application of the N B T test for such a purpose.

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Ten Patients with Early Hepatosplenomegaly

June 1975

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NITROBLUE TETRAZOLIUM T E S T

Material and Methods

90

80

70

60

50

r° 30

20

Controls

Non-osotic group

Ascitic group

FIG. 1. Percentage formation of formazan cells in the NBT test in two groups of splenomegalic bilharzial patients and in normal individuals (mean ± 1 S.D.).

tive of initial absolute granulocyte counts in subjects tested. Results T h e results of h e m a t o l o g i c a n d biochemical studies of the patients are listed in Table 1. Leukopenia, and particularly, deranged liver function tests, were more pronounced in the group with ascites. Nitroblue Tetrazolium Test T h e results of this test are listed in Table 2 and represented in Figure 1. Formazan cell formation by the PMN's of the patients who had hepatosplenomegaly and no ascites was not significantly different from that of the control individuals. Values of the NBT's of PMN's of patients who had ascites were significantly lower

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Subjects studied were men between 22 and 55 years of age hospitalized at AinShams Medical School. They all had hepatosplenomegaly whose schistosomal etiology had been confirmed by needle biopsy of the liver that showed portal fibrosis and schistosomal eggs surrounded by cellular reactions. The patients comprised two groups: (1) those who had hepatosplenomegaly and no ascites, and (2) those who had long-standing hepatosplenomegaly and ascites. T h e control individuals were men from the NAMRU-3 staff, of comparable ages but without a history of schistosomiasis. Results of screening tests consisting of complete blood counts, total serum proteins, albumin and globulins, serum bilirubin, SGPT and serum alkaline phosphatase were normal in these controls. Neither patients nor controls had any detectable bacterial infection at the time of sampling. T h e N B T test as a screen assay for bactericidal activity was applied on the PMN's of ten patients from each group and ten control subjects by the cytohistochemical m e t h o d of Gifford and Malawista,8 without the use of latex particles, as previously recommended by one of us. 22 In-vitro assessment of the bactericidal capacities of the PMN's from five individuals of each group was done by the method of Hirsch and Strauss 10 as modified by Quie and associates.17 Each assay was made simultaneously on washed PMN's from one patient and from one control subject, and on a blank medium containing bacteria without leukocytes. Normal pooled sera were used as a source of opsonins to exclude effects of complement fraction deficiencies in hepatic disorders. 12 T h e bacterial species used was Staphylococcus aureus, in a concentration of approximately 5 x 106 bacteria, and the ratio of PMN to bacteria was 1:1, irrespec-

N'itro blue tetrazolium test i00r

BAGNEID, KAMEL, AND SHAKER

924

A.J.C.P. —Vol. 63 Discussion

than those of both patients without ascites and normal persons (p < 0.001 for both).

Table 3. Phagocytosis and Bactericidal Effects on Staphylococcus aureus of Neutrophils in Schistosomal Hepatosplenomegaly and Ascites Viable Bacteria (x 106) Der ml. After 120 minutes Source of Polymorphonuclear Neutrophils

Time in Phagocytic Mixture (Minutes)

PMNfree Bacteria

PMNassociated Bacteria

Phagocytic Power*

Bactericidal Activityt

%

%

0

30

60

120

Patient 1 Control

4.4 5.6

2.20 0.60

0.90 0.20

0.80 0.10

0.24 0.05

0.60 0.05

95.5 99.1

81.8 98.2

Patient 2 Control

5.6 5.2

3.00 0.50

1.80 0.16

1.40 0.08

0.46 0.04

0.90 0.05

91.0 99.4

75.0 98.5

Patient 3 Control

3.0 1.0

1.60 0.24

9.94 0.08

0.84 0.04

0.30 0.02

0.60 0.02

92.0 98.0

72.0 96.0

Patient 4 Control

1.0 1.4

0.62 0.22

0.34 0.07

0.22 0.04

0.08 0.02

0.16 0.02

94.0 98.6

78.0 97.1

Patient 5 Control

1.9 1.4

0.96 0.18

0.80 0.07

0.75 0.04

0.24 0.02

0.51 0.02

87.3 98.8

60.5 97.4

Mean, five patients ± S.D.

3.18 ± 1.86

1.68 ±0.96

0.96 ± 0.52

0.81 ± 0.42

0.26 ± 0.14

0.45 ± 0.34

91.96 ± 3.14

73.46 ± 8.10

Mean, five controls ± S.D.

2.92 ±2.27

0.26 ± 0.21

0.12 ± 0.06

0.06 ±0.02

0.03 ±0.01

0.03 ±0.02

98.78 ± 0.53

97.44 ±0.99

* Phagocytic power = dead bacteria + leukocyte-associated viable bacteria at the end of incubation, expressed as percentage of initially incubated bacteria. t Bactericidal activity = dead bacteria after 2 hours' incubation, expressed as percent of initially incubated bacteria.

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Human intestinal schistosomiasis is a worldwide parasitic disease whose sequelae are hepatosplenomegaly with Phagocytosis and Bactericidal Capacity hepatic cirrhosis and, later, ascites. Late In these tests, leukocytes from patients hepatic disorders and the presence of who had no ascites showed no significant ascites in such patients are usually complidifference from those of normal controls. cated by frequent infections 21 and proTable 3 presents the numbers of viable tracted courses. 14 bacteria at different intervals of the exThe normal NBT's of patients who had periments and the phagocytic and bacte- hepatosplenomegaly without ascites point rial data for the ascitic patients. T h e to the integrity of their PMN's and expatterns of bactericidal activity at various plain the rarity of concurrent infections in intervals are illustrated in Figure 2. these early cases. This result could not be Phagocytosis and bactericidal capacities of related to the duration of infection as PMN's from these patients were sig- judged by the patients' ages and case nificantly impaired compared with those histories in an area where the incidence of of the normal persons (p < 0.01 and schistosomiasis in school children reaches p < 0.001, respectively). There was a posi- 100%. tive correlation between phagocytosis and T h e diminished host resistance to infecbactericidal activity of the PMN's. tions in patients who have schistosomiasis

June 1975 4 0

925

NITROBLUE TETRAZOLIUM TEST

r

30

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20

c Z a.

10

1

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L.H. Control

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M.K. Control

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30 60

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120

30 6 0

120

Incubotion time in minutes • Total vioble bacteria a WBC associated bocterio 0 Supernotont bocterio

Control bocterio (No WBC) Potient's WBC Control's WBC

FIG. 2. Phagocytosis and bactericidal capacity of neutrophils from five patients and five control subjects. Top row, increased numbers of viable bacteria after two hours' incubation, compared with their control neutrophils. Many of these bacteria are not associated with the leukocytes, indicating defective phagocytosis. Bottom row, the patterns and bactericidal capacities of the leukocytes of the patients and control individuals.

and hepatic cirrhosis and ascites has been suggested to result from chronic malnutrition, severe dysproteinemia, and changes in the immunologic processes, attributed possibly to inhibition of antibody synthesis and phagocytosis, or to inadequate immunologic properties of the globulins, which are increased in the disease. 2,7 T h e leukocytes, a major defense mechanism against infective agents, become reduced in number in intestinal schistosomiasis when splenomegaly supervenes. 7 The resulting leukopenia per se cannot, however, account for these frequent infections, especially since some cases are accompanied by leukocytosis.14 This investigation demonstrated dysfunction of the PMN's in schistosomiasis

with cirrhosis and ascites by the screening NBT histochemical test. This finding was substantiated by decreased bactericidal capacity, thus further supporting the validity of using the N B T test in such situations. The defective function of the PMN's demonstrated in this work is a contributory factor in the causation and/or the perpetuation of such infections. Our patients had no intercurrent infections and received no drugs known to affect the function of their leukocytes. 4,11 A combination of factors may be responsible for the diminished bactericidal capacity of the PMN demonstrated here: (1) Bacterial phagocytosis by PMN's in hepatic disease not including schis-

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BAGNE1D, KAMEL, AND SHAKER

References 1. Baehner RL, Nathan DG: Deficient glucose oxidation in the intact leukocytes of chronic granulomatous disease (abstr). Blood 28: 1010-1011, 1966 2. Bassily S, Higashi GI, Farid Z, et al: Serum immunoglobulins in schistosomiasis mansoni. J T r o p Med Hyg 75:73-75, 1972 3. Bittner SJ, Kieff E, Windhorst D, et al: T h e 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 60:843-853, 1973 4. Chretien J H , Garagusi VF: Suppressed reduction of nitroblue tetrazolium by polymorphonuclear neutrophils from patients receiving steroids. Experientia 27:1343, 1971 5. Concepcion VT, Rosner F, Feldman F: Nitroblue tetrazolium dye reduction in various hematologic disorders. NY State J Med 73: 952-956, 1973 6. Farid Z, Bassily S, Schulert AR, et al: Blood loss in chronic Schistosoma mansoni infection in Egyptian farmers. Trans R Soc T r o p Med Hyg 61:621-625, 1967 7. Farid Z, Prasad AS, Schulert AR, et al: Bilharzial splenomegaly. Arch Intern Med 113:37-41, 1964 8. Gifford RH, Malawista SE: A simple rapid micromethod for detecting chronic granulomatous disease of childhood. J Lab Clin Med 75:511-519, 1970

9. Gordon AM, Rowan RM, Brown T, et al: Routine application of the nitroblue tetrazolium test in the clinical laboratory. J Clin Pathol 26:52-56, 1973 10. Hirsch JG, Strauss BJ: Studies on heat-labile opsonin in rabbit serum. J Immunol 92: 145-154, 1964 11. Kaplan SS, Perillie PE, Finch SC: T h e effect of c h l o r a m p h e n i c o l o n h u m a n leukocyte phagocytosis a n d respiration. Proc Soc Exp Biol Med 103:839-843, 1969 12. Kourilsky O, Leroy C, Peltier A: Complement and liver cell function in 53 patients. Am J Med 55:783-790, 1973 13. Matula G, Paterson PY: Spontaneous in vitro reduction of nitroblue tetrazolium by neutrophils of adult patients with bacterial infection. N Engl J Med 285:311-317, 1971 14. Neves J, Martins NR: Long duration of septicaemia salmonellosis: 35 cases with 12 implicated species of Salmonella. Trans R Soc T r o p Med Hyg 61:541-552, 1967 15. Park BH, Fikrig SM, Smithwick EM: Infection and nitroblue-tetrazolium reduction by neutrophils: A diagnostic aid. Lancet 2:532-534, 1968 16. Pujol-Moix MN: Nitroblue-tetrazolium reducing capacity of neutrophils in diabetes. N Engl J Med 289:920, 1973 17. Quie PG, White JG, Holmes B, et al: In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46:668-679, 1967 18. Rudzka-Kantoch Z: Phagocytic activity of leukocytes in liver diseases. Arch Immunol T h e r Exp 16:709-721, 1968 19. Sbarra J, Jacobs AA, Strauss RR, et al: T h e biochemical and antimicrobial activities of phagocytizing cells. Am J Clin Nutr 24: 2 7 2 - 2 8 1 , 1971 20. Selvaraj RJ, Bhat KS: Metabolic and bactericidal activities of leukocytes in protein-calorie malnutrition. Am J Clin Nutr 25:166-174, 1972 22. Sherlock S: In Diseases of the Liver and Biliary System. Fourth edition. Oxford, Edinburgh, Blackwell Scientific Publications, 1968, p 405 23. Shousha S, Kamel K: Nitroblue tetrazolium test in children with kwashiorkor with a comment on the use of latex particles in the test. J Clin Pathol 25:494-497, 1972 23. Steigbigel RT, Johnson PK, Remington JS: T h e nitroblue tetrazolium reduction test versus conventional hematology in diagnosis of bacterial infection. N Engl J Med 2 9 0 : 2 3 5 238, 1974 24. Wollman MR, David DS, Brennan BL, et al: T h e nitroblue-tetrazolium test. Usefulness in detecting bacterial infections in uraemic and immunosuppressed renal transplant patients. Lancet 2:289-291, 1972

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tosomias was found to be diminished, especially when there was liver-cell necrosis. 18 T h e same factor(s) of hepatic dysfunction may be also operating in patients with l o n g - s t a n d i n g schistosomiasis a n d ascites who show significant disorders of hepatic function (Table 1). (2) This PMN defect may be due to iron and other nutritional deficiencies present in bilharzial patients 6 , 1 4 a n d known to interfere with PMN phagocytosis.19,20,22 T h e specific t r e a t m e n t s of schistosomiasis, vigilance for bacterial complications, bacteriologic surveillance, a n d appropriate antibiotic therapy, are all important in the management of late schistosomiasis. Our observations indicate that NBT screening is important in detecting leukocytic dysfunction in this disorder.

A.J.C.P.—Vol. 63

Effectiveness of the nitroblue tetrazolium test in demonstrating reduced bactericidal activity of polymorphonuclear neutrophils in schistosomal hepatosplenomegaly and ascites.

The nitroblue tetrazolium (NBT) test was carried out on blood from ten normal individuals and 20 schistosomal patients, ten of whom had hepatosplenome...
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