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1973

Use of the nitroblue tetrazolium dye test as an aid inmanaging patients with cystic fibrosisJames Francis SullivanYale University

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USE OF THE NITROBLUE TETRAZOLIUM DYE TEST AS AN AID IN MANAGING PATIENTS WITH CYSTIC FIBROSIS

James Francis Sullivan B.A. Manhattan College, 1969

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of Doctor of Medicine

Yale University School of Medicine 19 7 3

Acknowledgements

The author would like to express his gratitude and appreciation to the following individuals:

To my advisors, Dr. Thomas F. Dolan, Jr., and Dr. Robert H. Gifford for their friendly encouragement, advice and criticism during the preparation of my thesis and for the opportunity to work in their laboratories. Their friendship made the preparation of this thesis a worthwhile experience.

To Mrs. Kathleen Treat for expert technical assistance.

To Mrs. Dorothea McManamy and Miss Nancy Schwab for fine secretarial assistance.

Table of Contents.

Page

Introduction 1

NET False Positive Results 2

NET False Negative Reactions 5

Chronic Granulomatous Disease, 9

Phagocytosis, and Intracellular

Bactericidal Capacity

Theories on the Mechanism of NBT 15

Dye Reduction

Cystic Fibrosis 18

Application of the NBT Dye Test to 19

Cystic Fibrosis: Experimental

Proposals

Methods and Materials 22

Results 26

Discussion 32

Summary 39

Tables and Figures 40

Bibliography 50

Introduction

The white blood cell count is frequently used as a major

tool in the differential diagnosis of infectious diseases.

Although leukocytosis with a shift to the left is often indica¬

tive of bacterial infection, it is also known to accompany such

stressful situations as diabetic ketoacidosis, burns, strenuous

exercize, seizures, serum sickness, paroxysmal tachycardia,

salicylate or hydrocarbon poisoning, ether anesthesia, the post- 70

operative state, immunizations, hemolysis, and serious hemorrhage.

In his pioneer studies on the enzyme leukocyte alkaline phosphatase 98

(LAP), Wachstein discovered that the amount of LAP per neutrophil

and the number of LAP(+)neutrophiIs increased with bacterial

infection and he postulated that the increased enzyme activity

in neutrophils under inflammatory stimuli probably signified 92

increased cell metabolism. Ruttenburg demonstrated increased

LAP activity not only with bacterial infection, but also in

pregnancy, leukemoid reactions, and some cases of myeloid 10

metaplasia and polycythemia vera. Beisel discovered that LAP

rises witn some viral and rickettsial infections and that the

rises in LAP with bacterial or viral infection are often variable

in onset, sometime occurring several days after the fever and

leukocytosis have disappeared. Thus, changes in LAP have

proved to be of little value in the diagnosis of bacterial

infection.

A major step in the differentiation of bacterial infection

from those non-bacterial illnesses often confused with bacterial

infection because of fever, leukocytosis, and other clinical 80

symptoms has been made by Park and his associates with the use

of the nitroblue tetrazolium (NBT) dye test. These investigators

demonstrated that there is a significant difference in the per¬

centage of neutrophils reducing the dye to an intracellular blue-

black formazan precipitate (NBT(+)neutrophils) in patients with

active bacterial infection (mean 29-47%) vs. patients with viral

illnesses or other noninfectious fevers (including tuberculosis

and collagen diseases) and normal controls (mean 5.8-9.5%).

Their results were confirmed in a larger series of patients by 29,30 67,69

Feigin et.al. in children and Matula and Paterson in adults - 41,48.49,50,77

as well as in smaller series by other investigators. 29,30

In addition, Feigin's group, using discriminant analysis of the

percentage and absolute number of NBT (+) cells, were able to

construct a nomogram dividing their 349 febrile patients into

4 groups: 1) normal subjects, 2) individuals with viral infection,

noninfectious fevers, or partially treated bacterial infection,

3) untreated bacterial infections and 4) bacterial infection un¬

responsive to the therapy provided. In my thesis, I have applied

the NBT dye test to patients with cystic fibrosis in whom the

detection of active bacterial infection is often difficult. My

experimental proposals will be discussed later in the paper.

NBT False Positive Results

Despite significant support for the efficacy of the NBT

test in the differential diagnosis of bacterial from non-bacterial

2

disease, there have been cases of false positive and false

negative results.

A number of false positive NBT results have been reported

in the literature:

A) Parasitic Diseases - NBT results in the range usually

found in active bacterial infection (greater than 10% NBT (+) 67,69

neutrophils according to Matula and Paterson) have been found 2,18,67,69,87

in thirteen patients with malaria, in one patient each 18 18 18

with loiasis, trichinosis, and amebic liver absess and in three 28

dogs infected with Dirofilaria immitis (dog heartworm).

B) Fungal Diseases - high NBT values have been reported 80

in four patients with Candida albicans septicemia and in two 90

patients with pulmonary nocardiosis. 48

C) Viral and Mycoplasma Infections - Humbert reported

high NBT results in five out of twenty-five patients with viral

central nervous system infections (all five suspected or proven 26

Echovirus infections) while Elgefors reported elevated NBT

results in four out of eleven patients with viral meningitis - 32

one Coxsackie, two Herpes and one mumps meningitis. freeman

recently demonstrated high NBT results in two patients with

Mycoplasma pneumonia in which seroconversion was demonstrated. 82,83

D) Newborn and Premature Infants - Park et.al, 4 9 9 9 —

Humbert ejt. aM. , and Wehinger have demonstrated elevated NBT 80

responses in healthy term infants using the method of Park et.al. 37

(Park, Humbert), the method of Gifford and Malawista

3

(Wehinger) and a quantitative determination of NBT reduction 8

developed by Baehner and Nathan (Humbert). The elevated NBT 83

responses are said to return to normal levels in 7 - 20 days 25 82

or within two months. Park, Holmes and Good have demonstrated

an increased oxygen consumption, an increased hexose monophosphate

shunt activity and an increased quantitative NBT reduction in

the resting state of newborn infants and they interpret the

"false" positive NBT values as a result of increased metabolic

activity in the leukocyte.

Elevated NBT values were also found in the neutrophils of

healthy premature infants, and those with non-bacterial disease 19,20

by Cocchi et.al. who, in addition, found a decrease in NBT

reduction to "normal" levels (less than 10%) in premature infants

with bacterial infection.

E) Miscellaneous - False-positive NBT reduction reactions 39

have also been reported in Chediak-Higashi disease, after typhoid- 39

paratyphoid vaccine, in a case of suspected drug overdosage without 67 25 77

infection, in Hodgkin's disease with fever, in mvelofibrosis, and 48

in four out of six patients with hemophilia by Humbert et.al. 50

Humbert et.al. also reported elevated NBT reduction,leukocyte

respiration, and hexose monophosphate shunt activity in several

patients with ostenogenesis imperfecta, but these results are 14

disputed by Brown et.al. The concentration of heparin used in

80 the method of Park et.al has been reported to be a critical factor

4

23 determining the ranges of "normal"and has been reported to

41 elevate the level of spontaneous NBT reduction by neutrophils.

The critical role of heparin concentration has also been noted

in several of my experiments.

NBT False Negative Reactions

27 A) Bacterial Infections - Esposito and Lalla reported

twelve cases of bacterial meningitis in which the peripheral

blood neutrophils showed an NBT reduction response within the

normal range although the neutrophils within the cerebrospinal

fluid (CSF) showed a strongly positive NBT response. This is

in contrast to positive NBT reduction findings in peripheral 80

blood neutrophils in bacterial meningitis reported by Park ejt.al., 29,30 67,69 48

Feigin et.al,, Matula and Paterson, and Humbert et.al. 77

Ng reported three out of seven patients with subacute

bacterial endocarditis (SBE) had NBT reduction values by

neutrophils within the normal range in contrast to high NBT 84

reduction values reported by Park et.al.

Localized infections can also occasionally give negative

NBT responses as is the case with four patients reported by 69

Matula and Paterson with: staph>jLococcal osteomyelitis (presumably

after the hematogenous phase); cellulitis due to Group A beta-

hernolytic streptococci; urethritis-cystitis due to E. coli;

and an enterococcal pelvic absess.

Miscellaneous bacterial infections in which the NBT

5

response has been negative included a patient with nephrosis 78

and pneumococcal peritonitis cited by Park and three cases of 48

infected ventriculo-peritoneal shunts reported by Humbert et.al. 97 ~~

B) Drugs - Strauss et.al. found that phenylbutazone

inhibited both the engulfment and intracellular killing of E_^

coli by guinea pig polymorphonuclear leukocytes and also de¬

creased the quantitative reduction of NBT by neutrophils 57

stimulated with latex particles while Klebanoff and White

found that methimatazole inhibited the killing of Lactobacillus

acidophilus by the leukocytes of a patient with chronic granu¬

lomatous disease and the patient's mother's, but not by normal

leukocytes.

The effect of steroids on the NBT test is controversial. 73

Miller and Kaplan report that the quantitative NBT dye reduction

was decreased in twenty-one children receiving prednisone in a

dose of 1.0-2.0 mg/kg. compared to normal controls. Matula and 68

Paterson report one adult patient on 1 gram of methylprednisolone

and 150-200 mg. azathioprine daily with peritonitis and multiple

pelvic absesses in which the spontaneous NBT dye reduction by

neutrophils was negative, but the "stimulated" NBT test using 81

200 ug/ml of bacterial endotoxin described by Park and Good 77

was positive. Ng reported low NBT reduction and low "stimulated"

NBT tests with an undisclosed number of patients on steroids. 69

In contract to these results, Matula and Paterson, in another

paper, describe nine adult patients receiving the equivalent of

6

20-120 mg. of prednisone daily in which the NBT test was not

102 affected. In addition, a recent paper showed that the NBT

test was not affected by large doses of steroids and azathioprine

in renal transplant patients.

Similar controversy surrounds the effects of immunosuppressive

73

or cytotoxic drugs. Miller and Kaplan report normal NBT dye

reduction in leukemic children receiving 6-mercaptopurine and

48

amethopterin, while Humbert et.al report decreased NBT dye

reauction in two patients with septicemia on cytotoxic drugs

(histiocytosis X treated with vinblastine and lymphoma treated

62,63,64

with nitrogen mustard). Malawista demonstrated that

vinblastine does inhibit the increased oxygen consumption and

14

glucose -1- C oxidation that normally accompanies phagocytosis

and that it appears to limit lysosomal degranulation even though

the ingestion process itself and intracellular killing of bacteria

did not appear to be impaired, although a bacterial killing de-

65

feet was postulated in an earlier paper.

Sulfonamides and certain other anti-inflammatory drugs

found to be mydoperoxidase (an enzyme important in leukocyte

bactericidal activity) inhibitors may also result in a false

61

negative NBT test but apparently no patients have been tested

as yet.

C) Neutrophil metabolic defects/ inherited or acquired -

The major area where false negative NBT dye reduction

tests occur is with neutrophil metabolic defects or with some

impairment in host humoral or cellular defense mechanisms.

7

1) Inherited - The classic host defense disease

with a neutrophil defect in which the NBT dye test is negative 5,8,75,83.

is chronic granulomatous disease of childhood. The

importance of this disease in the development of the NBT test

and in the elucidation of the mechanism of phagocytosis and intra¬

cellular killing of bacteria will be discussed in the next 61

section. One patient with hereditary myeLoperoxidase deficiency

and a patient with complete deficiency of glucose-6-phosphate 22

dehydrogenase demonstrated defective bactericidal capacity and 78,84

a failure to reduce NBT. In addition, congenital agammaglobulinemia

has been reported to give negative results. A patient with

sickle cell disease and Salmonella osteomyelitis has been 78

reported to have a false negative NBT dye reduction test,

presumably due to defective polymorphonuclear leukocvte function 78

in this disease.

2) Acquired - Impaired neutrophil bactericidal capacity

was demonstrated with five test organisms and a decreased quan¬

titative NBT dye reduction test in a 75 year old patient with 24

mixed cryoglobulinemia by Douglas et.al, an acquired and

apparently transient, reversible condition. Certain of the 48

false negative NBT reduction group reported by Humbert et.al.

were postulated to have altered WBC function that may have

affected the results: two alcoholics, one with Pseudomonas

aeruginosa brain absesses and one with Streptococcus pyogenes

8

subdural empyema; a patient with severe burns and Pseudomonas

and Candida albicans septicemia; and one case of pneumonia 12

complicating an influenza syndrome. Bravton _et ._al. have shown

that alcohol depresses the rate of leukocyte mobilization

into traumatized skin of nutritionally normal people but human

polymorphonuclear leukocytes obtained after alcohol was given

or leukocytes exposed to alcohol in vitro showed no decrease

in ability to ingest or kill bacteria. This observation plus

the fact that no NBT dye reduction tests have been performed in

leukocytes exposed to alcohol must lead one to doubt alcohol as

a cause of the two false negative results in infected alcoholics. 1

Thermal Burns have been shown to affect neutrophil function

(phagocytosis and intracellular killing) and influenza virus

has been shown to reduce the phagocytosis of pneumococci by

exudate polymorphonuclear leukocytes, macrophaaes, and alveolar 93

macrophages in experimental animals. Both could plausibly

affect WBC function and thus account for false negative NBT

results.

Chronic Granulomatous Disease, Phagocytosis, and Intracellular Bactericidal Capacity

11 In 1957 Berendes, Bridges, and Good characterized the

features of a new syndrome, also elaborated by Landing and 59

Shirkey, which they later named fatal (chronic) granulomatous

13 disease of childhood. The syndrome consisted of lymphadenitis,

9

hepato-splenomegaly, and pulmonary infiltrates, pathologically

distinguished by granulomas which were clearly different from

other infectious and non-infectious granulomatous processes.

Patients reported with this syndrome have usually been males

with the onset of severe bacterial disease early in life.

Polymorphonuclear (PMN) leukocytes of patients with chronic

granulomatous disease of childhood (CGD) were noted to

phagocytize bacteria normally but to permit intracellular

survival of certain bacteria, along with a failure to reduce 45,75,89

NBT dye. These basic findings in PMN leukocytes of patients

with CGD stimulated work which elaborated not only the basic

features of this rare disease, but also some of the basic meta¬

bolic events associated with phagocytosis and intracellular

reduction and its intimate tie-in with these events.

The mechanisms of bacterial killing by polymorphonuclear

leukocytes are not completely understood. Phagocytosis of

bacteria is associated with the rupture of leukocyte granules

(lysosomes) and the discharge of their contents into the phago- 42,57

cytic vacuole containing the ingested organisms. Leukocytic

granules contain a variety of antibacterial agents among which 21,57 21

are lysozyme, a number of crranular cationic proteins and 94

myioperoxidase. There is also a burst of leukocyte metabolic

activity after phagocytosis that results in a sharp fall in 57,91

pH in the vicinity of the ingested particle, and in the generation

10

52,71,85 of hydrogen peroxide by the cell. Normally, these events

89

are accompanied by the death of most organisms. Quie et.al

demonstrated that the total lysozyme and phagocytin activity of

leukocyte extracts from patients with CGD were similar to control

leukocytes and they postulated that the deficiency of bactericidal

capacity of these leukocytes was due to the minimal degranulation

of lysosomes which they noted after phagocytosis. Further 43

studies demonstrated that PMN leukocytes of patients with CGD

responded to the phagocytosis of latex particles with normal

increments in glucose consumption, lactate production, Krebs cycle

activity, and lipid activity as do PMN leukocytes from normal 54,55

patients. These events were postulated to be associated with 43

the phagocytic process, a logical deduction since phagocytosis

is normal in both CGD and control leukocytes. However, unlike 52,54,55,85

normal leukocytes, the leukocytes of these patients

fail to show normal increments in respiration, direct oxidation

of glucose through the hexose monophosphate (HMP) shunt,

and hydrogen peroxide formation during particle uptake, events

thought to be associated with lysosomal degranulation and in- 43

tracellular bactericidal capacity. In normal leukocytes,

glycolytic inhibitors such as iodoacetate and fluoride inhibit

phagocytosis whereas respiratory inhibitors such as cyanide and

actinomycin A do not, indicating that the energy required for 97

engulfment comes from glycolysis. In addition, methylene blue

equally stimulated HMP activity in control and CGD leukocytes.

11

Therefore, the HMP pathway is present in CGD leukocytes, but 6

is not stimulated by phagocytosis.

A unified concept relating the various metabolic abnor¬

malities found in CGD has centered around the deficiency of

H2O2 in the leukocytes of these patients. Leukocytes of patients

with CGD can kill organisms which produce their own H90 2SUCH

53

as streptococci, (catalase negative) but they cannot kill organ¬

isms which produce catalase to destroy HO like Staphylococcus

57 60 22 or Serratia. Lehrer has demonstrated improved candidacidal

activity in CGD leukocytes with the use of the H2O2 producing

90 drug, phenazine. In addition, Reed has implicated H in the

stimulation of the HMP shunt and thus in CGD the deficiency of

H2O2 might also be responsible for the lack of stimulation of

58 72 HMP shunt. Klebanoff and McRipley and Sbarra demonstrated

the bactericidal potential of myeloperoxidase, iodine and H2O2

in the presence of bact.eria, thus demonstrating a major pathway

in which H?0? is used to kill bacteria intracellularly. 48

Klebanoff and White demonstrated that neutrophils from a patient

with CGD, unlike normal neutrophils, did not fix iodine after

the ingestion of heat killed Lactobacillus acidophilus or heat

killed or live Serratia marcescens♦ When live Lj_ acidophilus

was employed (produces H2O2), iodination did occur, although

it was less than normal. 6-8 4

Baehner and Nathan and Baehner and Karnovsky have found

a deficiency of a cyanide-insensitive NADH oxidase in the leukocytes

12

of patients with CGD, a finding they believe can account for

the metabolic abnormalities in their leukocytes. The postulated 90

mechanism is as follows:

2 NADH + 2H+ + 20^ --^2^-2xid§se-2 nad+ + 2 H2O2

2 H202 + 4 GSH _-.glutathione-2 GSSG + 4 H20

(reduced peroxidase (oxidized glutathione) glutathione)

2 GSSG + 2 NADPH + 2H+ —glutathione 2 NADP+ + 4 GSH reductase

A deficiency of NADH oxidase would thus account for a lack of

H202 and indirectly, since NADP+ is a factor known to control

HMP pathway activity, account for the lack of stimulation of

HMP shunt activity in the leukocytes of these patients. This 16

finding is supported by Cagan and Karnovsky who demonstrated

that the respiratory burst during phagocytosis in guinea pig

polymorphonuclear (PMN) leukocytes results from the action of

a cyanide-insensitive NADH oxidase, which produces H O 2 2 •

Further substantiation of this postulate was provided by Baehner, 5

Nathan, and Karnovsky who inserted an H2O2 generating system

into CGD leukocytes by allowing the cells to phagocytize latex

spherules coated with glucose oxidase. This produced amelioration

in the known metabolic deficiencies of these cells during

phagocytosis: a) formate oxidation dependent of H202 production

increased four times b) HMP shunt activity was markedly

stimulated and c) there was an increase towards normal levels

13

of NBT dye reduction of CGD leukocytes. However, these results 43

are contradicted by Holmes, Page and Good, who found that the

NADH oxidase activity of CGD leukocytes was equal to that of

normal control leukocytes.

Previously, I had mentioned that CGD is thought to be

primarily a sex-lined recessive found most often in males. 100

Evidence for this is provided by Windhorst, Holmes and Good

who found two populations of cells in PMN leukocytes of carrier

females. Leukocytes of all eight mothers and several sisters

of affected male patients were intermediate in bactericidal

capacity and quantitative NBT dye reduction between leukocytes

of affected boys and controls in line with the Lyon hypothesis. 8,57,75

This result has been verified by others. However, a few 8,88

cases of CGD have been reported in female children in which

no clinical or laboratory evidence of a leukocyte abnormality

(including normal NBT dye reduction) could be demonstrated in

family members, prompting an hypothesis that these cases could

be instances of autosomal recessive inheritance. Interestingly, 44

Holmes et.al. found that leukocyte glutathione peroxidase

activity was significantly diminished in two female patients

with CGD as compared to control leukocytes. By contrast,

leukocytes of male patients with CGD showed normal activity of 4 4

this enzyme. To support their position, Holmes ejt.al. have

cited the following postulated reactions to account for the

metabdic deficiencies of CGd leukocytes in their female patients

96 with glutathione peroxidase deficiency:

14

glutathione reductase--

GSSG + NADPH + H+ -

glucose-6-PO, + 2 NADP+ -^ME- H pathway

+ NADPH oxidase NADPH + H + 0n -

/L

glutathione H202 + 2 GSH -pefoxiaaii-

- 2 GSH + NADP +

2 NADPH + 2H+ + C02

- NADP+ + H202

GSSG + H20

In their scheme, a limitation in the pathway due to a deficiency

of glutathione peroxidase would block the cycle by limiting the

availability of GSSG (oxidized glutathione), the substrate for

glutathione reductase. Normal stimulation of the HMP pathway

would not occur, and oxygen consumption and hydrogen peroxide

production would be blocked owing to a lack of NADPH.

Thus two enzyme defects are postulated in CGD; an NADH

oxidase defect reported in both male and female CGD patients

and a glutathione peroxidase defect reported in certain female

but not male patients with CGD, each presented with theoretical

reactions which could account for the lack of H^O^ production

and the lack of stimulation of HMP shunt activity in the leukocytes

of these patients thus accounting for the bactericidal defect

and possibly for the mechanism of NBT reduction.

Theories on the Mechanism of NBT Dye Reduction

The major theory concerning the mechanism of increased 78

NBT dye reduction during bacterial infection assumes: 1) that

the phagocytosis system of the host, both cellular and humoral,

is operating normally and 2) that the infection involves the

15

This idea of the NBT test is based on systemic circulation. 80

the hypothesis that a metabolic change and an increased reduction

of NBT dye would take place when leukocytes are involved in vivo

during a natural infection. Oxidized (yellow) NBT dye in solu¬

tion does not penetrate cell membranes well, since it is positively 86

charged, therefore it is presumed that the NBT has to enter

the cell attached to a phagocytized particle or in the fluid 75

around it. The theory is that NBT dye reduction is increased

in bacterial infection as opposed to normal controls or nonin-

fectious fevers because contact with bacteria or bacterial 25,27

products stimulates phagocytosis and thus ingestion of the dye.

The increased intracellular metabolism within the polymorphonuclear

leukocyte, which is stimulated by phagocytosis, presumably is

also associated with the increased reduction of the oxidized, 78,80,81

yellow NBT dye to a reduced, blue-black formazan precipitate. 8

Baehner and Nathan, from their studies of CGD leukocytes, feel

that NADH oxidase is the intracellular enzyme responsible for

NBT dye reduction in the PMN leukocyte. The importance of

phagocytosis can be illustrated by the method of Gifford and 37

Malawista. Isolating granulocytes by allowing them to adhere

to a glass surface, these investigators found a high NBT re¬

duction in normal granulocytes. In the presence of infection

the percentage of NBT (+) cells did not increase, presumably

since the granulocytes were already "phagocytizing" glass and

thus maximally stimulated.

16

There is also experimental support that contact with

bacteria or bacterial products is responsible for the increased 20

NBT dye reduction during bacterial infection. Cocchi demonstrated

increased NBT dye reduction when polys were incubated with 81 69

Pseudomonas. Park and Good and Matula and Paterson also

showed stimulation of NBT dye reduction when the PMN leukocytes

were incubated with endotoxin. Staphylococcal protein-A and

streptolysin-0 can also induce an increased reduction of NBT 78

dye when they are added to whole blood, thus it is assumed that

any bacterial product or bacterial component may also be

responsible for the increased NBT dye reduction with bacterial

infection by stimulating increased phagocytosis and intracellular

reduction of the dye. 39

Crush feels that contact with bacteria or particulate

matter to stimulate the PMN leukocytes does not have to be

postulated and that lysosomal instability presumably can just

be the cause of increased NBT reduction. 38

Gifford and Malawista, in examining NBT dye reduction by

their method under different experimental conditions, concluded

that the permeability of the granulocyte to NBT may be the

critical variant and can be altered by changes in NBT concen¬

tration, particle ingestion, and adherence to a surface.

They postulated that reports which describe an increase or

decrease in NBT reduction associated with certain clinical dis¬

orders need not be interpreted to represent a stimulation or

17

depression of the enzyme system(s) necessary for NBT reduction

(or bactericidal capacity) as in CGD, but may be due, at least

in part, to change in cell permeability to NBT.

Cystic Fibrosis

Cystic Fibrosis is a hereditary disease of children,

adolescents, and young adults in which there is a generalized

dysfunction of exocrine glands. The triad of chronic pulmonary

disease, pancreatic insufficiency and abnormally high sweat

15,16,23

electrolytes is present in most patients. Hepatic

cirrhosis, intestinal obstruction edema, malabsorption, nasal

polyps, rectal prolapse and many other manifestations may be

part of the clinical picture of this protean disease. Most

of these symptoms are thought to be secondary to obstruction

of the organ passages (such as bronchioles, pancreatic ducts

23

and biliary ductules). There is general agreement that C.F.

is due to an inborn error of metabolism transmitted as an auto¬

somal recessive trait with an incidence of about 1 in 2000

live births in countries with populations predominantly of

76

Caucasian descent. The etiology of the disease is unknown,

but many studies have focused on the abnormally viscid mucus

as the key to understanding this disease. It has been suggested

34,35

by Gibson, Matthews and Minihan that a primary biological

function of mucus is to protect cell membranes from hydrostatic,

osmotic, and concentration gradients. The consequences predicted

18

for an impairment of this function (which would make mucus

"leaky" or "hyperpermeable") fit in with most of the known

characteristics of exocrine secretions from patients with

cystic fibrosis. Cystic fibrosis (C.F.) mucus was shown to

be hyperpermeable in vitro and serum from patients with C.F.

can induce hyperpermeability in vivo. Apart from its viscosity,

the most definitive abnormality of C.F. mucus is the quantitative,

but not qualitative difference between the electrophoretic

patterns produced by C.F. and normal submaxillary glycoprotein.

This difference seems entirely due to calcium since the addition

of calcium to normal mucus produces a C.F. pattern, while the

removal of calcium from C.F. mucus with editic acid produces a

40

normal pattern. This is especially interesting since probably

all C.F. exocrine secretions contain excess calcium. In the

34,35

studies by Gibson, Matthews, and Minihan, calcium was found

to increase mucus permeability in vitro and in. vivo and, when

iontophoresed into the skin, to raise the salt concentration

of sweat. They postulated that C.F. patients may have normal

mucus, but secrete an excess amount of calcium which combines

with the mucus and renders it hyperpermeable.

Application of the NBT Dye Test to Cystic Fibrosis:

Experimental Proposals

As was mentioned previously, C.F. patients are prone to

numerous pulmonary infections and it is usually respiratory

insufficiency secondary to chronic pulmonary disease that is the

19

cause of their demise. Infection developing distal to mucus

plugs in bronchioles is the most likely explanation for their

frequent infections, but a defect in these patients' poly¬

morphonuclear leukocytes has not yet been ruled out. It is

difficult to assess the presence or absence of acute bacterial

infection in these patients since pathogens are frequently

grown from the sputum of even asymptomatic patients and other

clinical parameters may be non-contributory. In addition, even

if antibiotic therapy is started for a presumed flareup of

lung disease, its effectiveness is difficult to assess since

often appropriate antibiotics may not change the patient's 36

sputum flora.

Since the recently introduced NBT dye reduction test has

been shown to be a useful guide in the diagnosis and management 29,30,48,69,80

cf bacterial infections, I decided to use the NBT

test in order to determine:

1) whether there was any difference in NBT dye reduction

by leukocytes of C.F. patients as compared to normal controls

as one method of determining the presence or absence of a

bactericidal defect in polymorphonuclear leukocytes of C.F.

patients.

2) whether the NBT test could be used as a useful parameter

in determining the presence or absence of an acute bacterial

infection in these patients.

20

3) whether the NBT test would be a useful guide to assess

the effectiveness of antibiotic therapy in these patients.

Other clinical parameters such as white blood cell count,

band shift, temperature, physical findings, sputum cultures

and chest X-ray results were also examined to determine their

effectiveness in predicting the presence or absence of bacterial

infection.

21

Methods and Materials

Patients

157 nitroblue tetrazolium (NBT) dye tests were performed

on heparinized venous blood obtained from 78 cystic fibrosis

(C.F.) patients seen at Yale-New Haven Hospital during the

period December 1971-May 1972. The diagnosis of cystic fibrosis

was established in these patients by means of elevated sweat

33 95

chloride determinations and a typical clinical picture. The

patients ranged in age from 7 months to 26 1/2 years with a

mean of 8.6 years.

At each visit, the patients were placed into one of five

95

groups on the basis of the Shwachman Chest X-ray score (see

Table 8) and the clinical assessment of the senior physician

on the basis of history, physical examination, chest x-ray,

and other laboratory parameters if available. The results of

the NBT dye test were not known at the time the categorization

was made and the clinical assessment was not known to the person

reading the results of the NBT test.

The groupings were as follows: Group I patients had

Shwachman Chest X-ray scores greater than 19 and were stable

and doing well. Group II patients also had Shwachman Chest X-ray

scores greater than 19 but were thought to have an acute flareup

of their disease with an increase in respiratory symptomatology

and a pulmonic process thought to be bacterial. Group III

22

patients had more chronic changes in their lungs with Shwachman

Chest X-ray scores less than 19 but were thought to be stable

and doing well, on or off antibiotics. Group IV patients

had Shwachman Chest X-ray scores less than 19 but were thought

to have an acute flareup of their disease with an increase in

respiratory symptomatology and a pulmonic process thought to be

bacterial. Group V patients were a small, difficult group to

clinically define with Shwachman Chest X-ray scores less than

19 who were thought to be chronically infected and gang gradually

downhill without ever going back to a stable baseline - con¬

tinuously doing poorly with exercize intolerance, dyspnea or

tachypnea, bouts of hemoptysis, copious sputum production, poor

A pulmonary function tests, with rales and rhonchi consistently

heard in their chest and on continuous antibiotic therapy

without amelioration of symptoms.

I also analyzed my results by dividing the patients into

those judged clinically well (Groups I and III) , those judged

clinically sick (Groups II and IV) and those judged to be

chronically infected (Group V).

I also compared the results of the NBT test with other

clinical parameters - white blood cell count, band shift (shift

to the left in the polymorphonuclear (PMN) leukocyte series),

A temperature, the presence or absence of rales, sputum culture

results and findings on chest x-ray to see how good an indicator

23

each might be in predicting the presence or absence of a

bacterial infection.

Laboratory Methods

The NBT dye test was performed according to the method 80

of Park et.al with a slight modification. One cc of venous

blood was drawn into a plastic syringe and transferred to a

disposable plastic tube to which 100 units of heparin were added.

At the same time, blood was drawn for a white blood cell count

and differential count to be performed by the Yale-New Haven

Hospital Hematology Lab, or, in certain instances, by myself.

Approximately 0.1 ml. of this heparinized blood was then trans¬

ferred into a clean, concave microslid.e (Macalaster-Bicknell

Co., New Haven, Conn.) and mixed with an equal amount of NBT

solution (a mixture of equal amounts of 0.2% NBT (Nutritional

Biochemicals Corp., Cleveland) in physiological saline solution

and 0.15 M phosphate-buffered saline solution - pH 7.2). The

slides were placed in petri dishes containing wet gauze to

provide humidity and were then incubated at 37°C. for 15 minutes

and subsequently kept at room temperature for an additional 15

minutes. At the end of this period, the blood-NBT mixture was

again mixed using a small wooden applicator stick. Coverslip

smears were carefully made, air dried, and counterstained with

Wright's stain. The tests were all performed within 1/2 hour

of obtaining the blood samples.

24

Cell Count

Wright stained smears were examined under the microscope

with the high, dry objective (450X). Initially three separate

counts of 100 neutrophils each was performed on each test but

since a good correlation was obtained on each of the counts,

only 100 neutrophils were counted for the majority of tests.

Monocytes were not counted. Only those cells which were intact

and could be clearly identified as neutrophils with a discrete

deposit of blue-black formazan were classified at NET positive.

The intracellular formazan deposit could be either in the form

of a "clump" or a "streak" of blue-black granules. The absolute

number of positive neutrophils per cu. mm. was calculated

using the white blood cell count, differential, and the percentage

of NET (+) neutrophils. In our study, as well as

scores above 10% were considered abnormal, or NBT

correlating with the presence of active bacterial

NBT scores below 10% were considered to be in the

but were also seen in patients with viral disease

infectious febrile disease.

69 others, NBT

positive,

infection.

normal range,

or other non-

25

Results

131 tests on 78 separate patients with cystic fibrosis

were classified into one of five groups according to the criteria

discussed earlier. Twenty-six tests were not classified into

one of the five groups because they were part of sequential

studies taken while the patients were on antibiotics for a

presumed flareup of respiratory infection and will be discussed

separately.

Table 1 shows the results of the one hundred thirty-one

tests broken down according to the five groups. The results

are also analyzed as to the number of tests in each group that

had less than or equal to 10% of the neutrophils reducing the

dye and those that had greater than 10% of the neutrophils

reducing the dye. The results of my scatter plot of values

(Figure 2) indicated that 10% NBT ( + ) neutrophils would be a

good cutoff point in my series. Patients placed in Group I

had an age range from 2-19 1/2 years and had a mean percentage

of NBT ( + ) neutrophils of 7.4 + 5.1% with a range of 1-32%.

Thirty-eight tests had an NBT score less than or equal to 10%

and four had an NBT score greater than 10% which is statistically

significant (p^.QOl). Of the four patients with NBT scores

^10%, two values (11% and 32%) were obtained on two separate

occasions from one patient thought to be clinically healthy;

another value of 11% on a clinically healthy patient and a

value of 20% on a patient thought to be stable at the time

of his clinic visit but who spiked a temperature to 102°F.

26

that night with increased cough and sputum production.

Patients placed in Group II with a presumed flareup of

respiratory infection had an age range from 7 months to 18

years and had a mean percentage of NET (+) neutrophils of

23.1 + 11.3% with a range of 9-56%. Eighteen tests had an

NBT score of greater than 10% and one test (9%) had an NBT

score less than or equal to 10%, which is statistically

significant (p<.001).

Patients placed into Group III had an age range from

1-18 years with a mean percentage of NBT (+) neutrophils of

6.3 +2.5% with a range of 2-14%. Forty tests had an NBT

score of <10% and two tests (11% and 14% on two separate

patients) had an NBT score of greater than 10% which is

statistically significant (p<.0Ql).

Group IV patients with a presumed flareup of respiratory

infection had an age range from 3-26 1/2 years with a mean

percentage of NBT ( + ) neutrophils of 17.4 + 5.6% with a range

of 4-27%. Nineteen tests had an NBT score of greater than 10%

and two tests (8% and 4% on two separate patients) had an NBT

score of ~bl0%, which is statistically significant (p<.001).

Group V contained a small number of patients wth an

age range from 5-19 1/2 years with a mean percentage of NBT (+)

neutrophils of 9.4 + 3.4% with a range of 5-15%. Five tests

had an NBT score of <10% and two tests had an NBT score greater

27

than 10%, which was not significant.

Table 2 analyzes the results of the NBT percentage

breakdown according to whether the patient was thought to be

clinically infected (Groups II and IV), clinically stable

(Groups I and III), or chronically infected (Group V). In

those patients thought to be clinically stable (84 tests on

62 patients) , seventy-eight tests (93%) had an NBT score of

10% and six tests (7%) had an NBT score greater than 10%,

which is statistically significant (p.<1.001). In those

patients thought to be clinically infected (40 tests on 34

patients), thirty-seven tests (93%) had an NBT score of greater

than 10% and three (7%) had an NBT score of £-10%, which is

statistically significant (p<.0Ql). Five tests in those

patients thought to be chronically infected had an NBT score

of £10% and two tests had an NBT score of greater than 10%,

whichis not statistically significant.

Table 3 shows the results of the mean percentage of NBT (+)

neutrophils, the mean absolute number of NBT (+) neutrophils,

the mean white cell count, the mean percent band forms, the mean

temperature and the change in sputum flora in those patients

thought to be clinically stable (Groups I and III) and those

thought to be clinically infected (Groups II and IV). These

same parameters were also analyzed in Table 4 according to the

five clinical groups. The mean percentage of NBT (+) neutrophils

28

'

based on 84 tests on 62 clinically stable patients was 6.8 +4.0%

while the mean NBT % in 40 tests on 34 clinically infected

patients was 20.1 + 9.1%, which is a statistically significant

difference (p<.01). The mean absolute number of NBT (+)

neutrophils is also statistically significant (p^.001) between

the two groups, the mean of those clinically stable being

448.2 + 330.8 and the mean of those thought to be clinically

infected of 1412.9 + 1263.8. The mean white blood cell count

(11,500 + 3,400 vs. 11,500 + 4400), band shift (2.5 + 3.6 vs.

4.9 + 6.7) and temperature (98.8oFe + 0.8 vs. 99.8oF* + 1.5)

were not statistically significant between the clinically

infected and clinically not infected groups.

Other clinical parameters were analyzed in these patients.

Table 5 shows the results of sputum cultures taken on these

patients broken down into the type of organism isolated for

each of the five groups and whether there was any change in the

organism(s) isolated from the most recent culture. Only Group I

has a significant number of sputum cultures with normal flora

(21 out of 42) while Pseudomonas appears to be the most commonly

isolated organism, increasing in proportion as we go from

Group I to Group V. Staphylococci and coliform organisms are

less frequently isolated and Hemophilus influenza is rarely

isolated in our clinic. There was not much difference between

the first four groups in the proportion of cultures in which

there was a change in the organism(s) isolated from the previous

I 29

culture: 20/42 in Group I; 8/19 in Group II; 17/42 in Group III

and 9/21 in Group IV. In Group V only one out of seven cultures

showed a change in the organism(s) isolated.

Table 6 depicts the presence or absence of a change in

the chest x-ray in each of the five groups. In most instances,

chest x-rays were not done on that particular day, but in the

few that were, a new infiltrate was found in 4 out of 5 films

taken in Group II patients but in only 3 out of 9 of the patients

in Group IV. In the clinically stable groups, Group I had no

films with a new infiltrate and Group III had a change in 4 out

of 15 films. In Table 7 rales were found to be present in 9

and absent in 56 of those patients thought to be clinically

stable and to be present in 18 and absent in 12 thought to be

clinically sick.

Sequential Studies

Fourteen patients have been serially followed with NBT

examinations during 16 bouts of presumed bacterial pneumonitis.

Prior to their flareup in respiratory symptoms, each patient

had a "normal" value of £10%NBT (+) neutrophils. With the onset

of the presumed acute bacterial pneumonia, the percentage of

NBT (+) neutrophils rose into the "infected" range of greater

than 10% in all the patients studied.

In 11 instances of presumed bacterial infection, the

institution of antibiotic therapy "appropriate" for the patient's

30

sputum flora was followed by a prompt resolution in clinical

symptomatology and a return of the NBT to "normal" levels.

In two instances the NBT test remained elevated and the patients

remained symptomatic until an empiric change in antibiotics was

instituted, whereupon clinical improvement coincided with a

decrease in NBT test to "normal" levels. In three patients,

the NBT test did not return to normal and the clinical condition

deteriorated in these cases. Figure 1 illustrates the response

of the NBT test to antibiotic therapy in two patients, one of

whom (pt. B) had a prompt clinical response and a return of

the NBT to "normal" levels and one patient (pt. A) just

previously mentioned who failed to show a clinical response to

antibiotic therapy and eventually expired and whose NBT test

remained elevated.

31

Discussion

Seventy-eight patients with cystic fibrosis of the

pancreas were studied prospectively for six months to see if

the NBT test would be of value in evaluating these children.

The results of the NBT test in these patients provide useful

answers to the three experimental proposals previously elaborated:

1) The results of the NBT dye test indicate that the

leukocytes of patients with cystic fibrosis reduce the dye in

the same relative proportions as do the leukocytes of normal

control patients with or without bacterial infection. The

mean percentage of neutrophils reducing the NBT dye in our

patients with cystic fibrosis who were clinically stable

(Groups I & III) was 6.8 + 4.0%, which is in no way different

from normal, healthy controls without cystic fibrosis either

29,30,48,69,80

in our laboratory (5.1 + 3.6%), or as reported by others.

The positive NBT tests (mean 20.1 + 9.1%) in our patients with

cystic fibrosis with suspected bacterial infection of the

respiratory tract was similar to control patients with

bacterial infection both in our lab (20.0 + 6.9%) and as

2,29,30,48,67,69,80

reported by others.

2) The results indicate that the NBT test is a very

useful parameter in determining the presence or absence of

suspected acute bacterial infection in these patients. An

NBT test result with <10% of the patient's neutrophils reducing

the dye was correlated in 78 out of 84 instances with a patient

32

thought to be clinically stable (Groups I & III) while an NBT

test result of^>10% of the patient's neutrophils reducing the

dye was correlated in 37 out of 40 instances with the diagnosis

of probable acute bacterial respiratory infection (Groups II &

IV). It is significant in looking at our six false positive

results that one (20%) was in a patient thought to be clinically

stable at his clinic visit in the afternoon, but who developed

a temperature of 102°F. with increased cough and sputum production

that night and that our highest false positive value by far (32%)

was in a girl who had elevated values (11% and 32%) on two

separate occasions without any evidence of acute bacterial

disease. The results of the NBT test in our group of chronically

infected patients (Group V) shows a random distribution around

the 10% cutoff point. This result is not too surprising when

one considers that these patients are more or less continuously

infected and are probably intermittently having acute invasion

of bacteria across bronchial walls. Alternate explanations

include possible metabolic deficiencies in their leukocytes

induced by long-standing disease or by prolonged antibiotic

therapy. The NBT test appears to be particularly helpful in

patients with chest x-ray scores under 19 (Groups III, IV, V)

since this group of patients frequently have Pseudomonas as

the predominant flora (see Table 5), and evidence of an acute

infection requires replacement of an oral antibiotic with

parental antibiotics such as Gentamicin, Colymicin, or 46

Carbenicillin.

3) Results on a limited number of patients (16 instances

of probable bacterial infection in 14 patients) indicate that

the NBT test appears to be a useful guide in assessing the

effectiveness of antibiotic therapy in patients with cystic

fibrosis. In each of the 14 patients a normal baseline NBT

value became elevated into the positive range ( >10%) with the

onset of a probable acute bacterial infection. In 11 instances,

the institution of appropriate antibiotic therapy was followed

by prompt resolution of the clinical signs and symptoms and was

accompanied by a decrease in the NBT test to normal levels. In

two instances the NBT test remained elevated and the patients

remained symptomatic until a change in antibiotic therapy was

instituted, whereupon clinical improvement and a decrease in

the NBT results to normal levels occurred. In two instances the

patients showed a poor response to antibiotic therapy and their

NBT values remained elevated. One patient died and the other

was discharged from the hospital with little clinical improvement.

Another terminal patient with cystic fibrosis was admitted to

the hospital and put on an antibiotic regimen which resulted

in the fall in his NBT test to normal levels within three days

although he expired later that third day. Whether his therapy

was effective in treating his probable bacterial pneumonia and

34

his death secondary to chronic pulmonary insufficiency with

hy;oxia and cardiorespiratory arrest or whether this was a

false negative result is unknown.

Also significant was my analysis of other clinical

parameters usually helpful in the diagnosis of bacterial disease.

The mean white blood cell count, the mean percent band forms,

the mean temperature and the number of patients with a change

in sputum flora from previous culture were not significantly

different between those patients thought to be clinically stable

(Groups I & III) and those patients (Groups II & IV) thought to

be clinically infected (Tables 3 & 4). The presence or absence

of rales (Table 7) was not a consistent help in differentiating

those thought to be clinically sick from those thought to be

clinically stable. In a limited number of studies, the presence

or absence of a new infiltrate on chest x-ray (Table 6) was

helpful only in differentiating Group I patients (10 out of 10

showed no change on chest x-ray) from Group II patients (4 out

of 5 showed a new infiltrate on chest films). The chest x-ray

did not appear to be useful in differentiating Groups III, IV,

or V most probably because chronic changes in the lungs of these

patients tend to obscure the presence of a new infiltrate.

Sputum culture results (Table 5) indicate that only Group I

has a significant number of cultures with normal flora (21 out

of 42) while Pseudomonas appears to be the most commonly

35

isolated organism, increasing in proportion as we go from Group I

to Group V. That Pseudomonas appears to be the most commonly

isolated organism in our series, with less isolates of

Staphylococcus and coliform organisms and very few isolates of 47,51,56

Hemophilus influenza is in disagreement with other studies

in which the Staphylococcus is the most common pathogen isolated

followed by Pseudomonas, H. influenza and the coliforms. Also

significant was the fact that there was not much difference

between the first four groups in the proportion of cultures

in which there was a change in the organism(s) isolated from

the previous study. Thus, the classical rule of isolating the

pathogen and treating with appropriate antibiotics is clouded

in patients with cystic fibrosis in which acute flareups of

bacterial infection may not be reflected by changes in their

sputum flora. In addition, the classical rule of treating

the infection until the pathogen disappears and clinical

improvement occurs also does not complete^hold in patients

with cystic fibrosis in which appropriate antibiotic therapy

may not change the sputum flora (probably because effective

concentrations of the antibiotic cannot penetrate into the 6 6

sputum). In our patients clinical improvement and the

return of the NET test to normal after antibiotic therapy was

frequently not accompanied by a change in sputum flora.

In my study, the absolute number of NBT (+) neutrophils

was also statistically significant (p <.001) between those

patients in Groups I & III thought to be clinically stable

36

(448.2 + 330.8/cu. mm.) and those patients in Groups II & IV

thought to be clinically infected (1412.9 + 1263.8/cu. mm.)

(see Table 3). However, in analyzing the scatter plot of

values (Figure 2) it was decided that a cutoff of 10% NBT (+)

neutrophils would be both a simpler and more reliable test.

In addition, a simple analysis of the scatter plot indicates

that discriminant analysis using the percentage of NBT (+)

neutrophils and the absolute number of NBT (+) neutrophils 29,30

per cu. mm. in the style of Feigin's nomogram would not be

more beneficial than the simple cutoff of 10% NBT (+) neutrophils.

One reason to account for the failure of discriminant analysis

to be beneficial in examining our results is the failure of the

white blood cell count to increase significantly with infection

in our patients with cystic fibrosis as compared to most normal

control patients with infection.

An important objection to this study is that the significance

of the NBT test has been assessed by clinical judgment together

with other available laboratory data. The clinical parameters

which usually indicate bacterial infection can be misleading

in patients with cystic fibrosis as discussed earlier. At

times, the only symptoms of an acute flareup of respiratory

disease might be an increased cough. It is also frequently

impossible to know if an increase in symptomatology in these

patients harboring respiratory pathogens indeed have a bacterial

37

or viral infection as a cause on many occasions. For this

reason, our patients thought to be clinically infected with a

probable bacterial infection had this diagnosis made primarily

on the judgment of the senior clinician in the cystic fibrosis

clinic. Secondary supporting evidence for the diagnosis was

made in most cases by the response of the "probable" bacterial

infections to antibiotic therapy or to a change in antibiotic

therapy, which resulted in a prompt decrease in respiratory

symptomatology. There is also abundant evidence, based mainly 80 29,30

on studies by Park et.alL, Feigin et.al_ and Matula and 67,69

Paterson and from our laboratory that a high NET test

correlates very well with the presence of bacterial infection

and there is abundant evidence that patients with cystic

fibrosis develop recurrent infections in the lower respiratory

tract.

I would conclude, based upon the data presented, that

the NBT test is a valuable parameter, along with a careful

history, physical examination, and other laboratory data in

the surveillance and management of patients with cystic fibrosis.

38

Summary

One hundred fifty seven nitroblue tetrazolium (NBT) dye

tests were performed on seventy-eight patients with cystic

fibrosis. A "positive" NBT response of > 10% was significantly

correlated with the presence of presumed bacterial respiratory

infection and stable patients had "normal" NBT scores below

10%. Serial determinations in sixteen instances of bacterial

infection were helpful in evaluating response to therapy.

Total leukocyte count, mean percentage of immature polymorphonu¬

clear leukocytes, temperature or a change in sputum flora were

not helpful in detecting bacterial infection in these patients.

The NBT test is a valuable adjunct in the surveillance

of patients with cystic fibrosis.

39

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Table 7

Correlation of the presence or absence of rales with the

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Clinically Well

(Groups I & III)

Clinically Sick

(Groups II & IV)

Chronically Infected

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Rales

Present 9 18 6

Rale s

Absent 56 12 4

Chest x-ray score Chest x-ray score

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58

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