Original Paper Vox Sang 1992;63:4&52
Piero Borzini Giorpio Assali CentrYu Trasfusionale e Laboratorio di Ematologia, Ospedale San Gerardo, Monza, Italia
Granulocyte Serology Findings in Juvenile Symptomatic ldiopathic Autoimmune Neutropenia Using a Multiassay Procedure Report on 21 Cases
................................................................................................. Abstract The serological findings on 21 children with idiopathic autoimmune neutropenia are reported. A multiassay procedure was adopted including agglutination, immunofluorescence and cytotoxicity tests. Beside a cause-effect correlation between granulocyte antibodies and clinical course, a serologic polymorphism was found. As the sensitivity of each assay seemed to be related to the characteristics of the involved antigens and antibodies, as well as to the intrinsic sensitivity of the tests, the performance of a multiassay procedure appears to be advisable for the diagnosis and the follow-up of autoimmune neutropenia.
.....................
Introduction
Juvenile idiopathic autoimmune neutropenia is a welldocumented, although uncommon, children's disease [1-4]. Children are affected mainly during the first 2 years of life [ 5 ] ;the clinical course is generally benign with spontaneous resolution within 6-24 months from onset, without sequelae [5-71. The neutropenia is very often associated with the presence of auto- and alloreactive neutrophil-specific antibodies; most of these antibodies recognize polymorphic alloantigens and the immunopathogenetic origin of immunization is still unclear [4,8, 91. We report the serologic findings of 21 children with symptomatic idiopathic neutropenia. Findings were quite uniform and in good agreement with the clinical condi-
Received: Jan. 18,1991 Revised manuscript received: Nov. 25. 1991 Accepted: Nov. 27, 1991
tion. Some differences in the reactivity of the serum antibodies with the target cells were consistent with an immunologic polymorphism of the disease.
Materials and Methods Patients Twenty-one patients were investigated. The patients' blood samples were referred to our laboratory from the pediatric divisions of eight different hospitals. In all cases the peripheral blood smears were observed, while wecouldexamine the bone marrow specimens in only one third of the patients (those referred from our hospital). At the time of the first observation, 16 patients were symptomatic while 5 were free of symptoms and probably entering the recovery phase.
Dr. Piero Borzini Centro Trasfusionalee Lahoratorio di Ematologia Ospedale San Gerardo Via Snlferino. I6 20052 Monza (Italy)
0 1992 S. Karger AG. Basel 0042-90071921Oh314W8 $2.7510
Serologic Investigations The following investigations were performed: the indirect granulocyte suspensionimmunofluorescence test (GIFT), the leukoagglutination test (LAT), the granulocyte agglutination test (GAT), the granulocyte cytotoxicity test (GCT), and the lymphocytotoxicity test (LCT). The target cells were obtained from a panel of at least twelve random (0 blood group) blood donors. The NA1 specificity was determined using a panel of 6 donors with known NA1 phenotype. Target Cells Leukocytes to be used as target cells for the LAT were isolated from blood collected in NazEDTA (10 mM final concentration). One milliliterofdextran6% (w/v) wasaddedto5 mlofwholeanticoagulated blood and the volume was brought to 10 ml with 0.15 Mphosphatebuffered saline (PBS) pH 7.2. The tubes were incubated for 30 min at 37°C. The supernatant was then collected and centrifuged. The cells obtained were washed three times with PBS containing 8 mM EDTA (PBS-EDTA). After the last wash the contaminating erythrocytes were submitted to hypotonic lysis with a short incubation (40 s) with 33 mM NaCI. The leukocytes were washed three times with PBSEDTA containing 0.3% bovine serum albumin (PBS-EDTA-BSA). The granulocytes to be used for the granulocyte agglutination test (GAT) andfortheGIFTwereobtained asfollows. Theleukocyte-rich suspension was obtained as described above, and the lymphocytes were removed by gradient sedimentation (d = 1.077).The neutrophils were collected from the bottom of the tubes. After washings with PBS-EDTA-BSA, they were resuspended in the same buffer and used to perform GAT. A portion of the granulocyte suspension was fixed with 1%paraformaldehyde solution, pH 7.2, for5 min at 22°C. After three washings with PBS-EDTA-BSA, they were used to perform GIFT. Lymphocytes and granulocytes for the cytotoxicity assays (LCT and GCT) were obtained by dextran sedimentation followed by gradient sedimentation, as described above, using blood collected in heparin (1 unit/ml) and using RPMI-1640 with Hepes, containing20Y0 fetal calf serum (FCS), as working buffer (RPMI-FCS). Assays GIFT GIFT was performed on paraformaldehyde-fixed granulocytes [lo]. The fixed washed cells were incubated with patients' sera for30 min at 37"Cfor IgG detection and at room temperature for IgM detection. Class-specificFITCF(ab')2antihuman IgG and antihuman IgM were used for isotype characterization. GATand LAT GAT and LAT were performed using unfixed cells [lo, 111,incubating 50 pl of cell suspension (at 3-5 x lO'/ml) with 100 111 of serum, for 2 h. LCT and GC?: LCT and GCT were performed in 60 microwell tissue-typing trays [12,13]. The cells were resuspended in RPMI-FCS and brought to 4-6 x 106/ml.LCT was performed using standard NIH conditions, while, for GCT, the complement-mediated lysis was prolonged for 3 h. Scoring The positive reactions were scored as follows: agglutination and immunofluorescence tests: from t w to t t (subjective evaluation); cytotoxicity asssays: from 2 to 8 (according to the percentage of cell death).
+
ResuIts
The mean age of the 21 patients was 16.4 months (range 9-40, median 14). Sixteen patients entered the study in a symptomatic phase of the disease and 5 were subjected to a unique serological investigation some months after symptomatic recovery. All patients considered together had a median of 8 infections per year during the symptomatic course of their disease. A great variety of muco-epidermal infections sustained by a variety of gram-positive and gram-negative bacteria were reported. Curative antibiotic treatment and highdose intravenous gammaglobulins were administered during the acute phase of the disease. Chronic phases were variously treated: in most cases, prednisone (1 mgl kg/day) and prophylactic antibiotics were administered when the neutrophil count was below 500/p1. No patient received leukocyte transfusions. The leukocyte count was in a wide range (1,100-18,000) while the neutrophil count was markedly low in the symptomatic patients (range 0-650/p1; mean 400; median 500) in comparison with the asymptomatic patients (range 1,000-2,100; mean 1,400; median 1,200). The differential leukocyte count of the peripheral blood was characterized by the presence of a discrete percentage (5-20%) of mononuclear cells deriving from the myeloid series (myelocytes and degranulated metamyelocytes). In 7 symptomatic cases, the observation of bone marrow specimens revealed myeloid hyperplasia, the absence of mature and hypersegmented neutrophil, and an arrest of maturation at myelocyte or metamyelocyte stage. Table 1represents the serological findings of the 39 sera from the 21 patients studied. No antibody was found in 5 patients (23%). These patients are listed at the end of the table: all of them were free of symptoms and their neutrophil count was within the normal values or borderline. Sixteen patients (77%) were serologically positive: 6 out 34 sera from these patients were negative at the last observation. These patients (cases 4-6, 10 and 12) were considered clinically cured at the time of the last observation. Two other patients (cases 3 and 8) were clinically cured at the time of the last observation: their sera were still reactive in the agglutination assay, while the peripheral neutrophil count was within the normal range. One interesting technical finding was the evidence of variable scores of the positive reactions obtained with the different donor's target granulocytes tested with the same sera. This finding will be discussed later.
49
Table 1. Age, observation date, neutrophil count per microliter and serological findings in 21 children affected with idiopathic juvenile autoimmune neutropenia
Case
Age months
Date
PMN
GAT
13 21 15
2/88 11/88 1/88 1/89 2/88 4/88 6/88 12/88 3/88 4/88 5/88 7/88 1/89 4/88 5/88 6/88 12/88 6/88 7/88 7/89 11/89 12/89 4/89 4/89 2/90 10190 11/90 12/89 1/90 2/90 2/90 3/90 12/90 12/90 3/88 5/88 1/89 7/89 1/90
150 500 500 600 50 50 550 950 650 600 1100 1750 1650 650 750 900 1200 500 500 450 650 650 0 350 2000 300 800 400 850 1000 80 550 6.50 650 1200 1500 1000 2100 1200
+w/+
LAT
GIFT
+w/++
+W/+t
GCT
LCT
Percent Ig class of panel
Specificity
-
1 2 3
4
5
6
7 8
16
12
13
14 24
9 10
12 15
11
15
12 13 14 15 16 17 18 19 20 21
20 12 39 10 10 9 40 17 9 9
++
+W
-
CW
+W
+W
+W
+W
-
+ + / + + t++I+++ +I+++ +I+ t
+
+w/++ +w/+t
50
NI NI NI NA 1
+W
+W
-
-
-
+I+
+
-
-
+
100
NI
25
NI
20
NI
50
NAI
50 60
NA 1 NI
+W
-
-
+
-
+W +W
-
+ + +/+ + + +W +++
+W +W
t +W +W
-
+I+++ -
+++ +W
+/++
+
+W
-
++ +W +
+W
-
-
-
+++ +
+++
+W
+W
+
+W
-
-
-
-
-
-
-
-
-
-
PMN = Neutrophil polymorphonuclear cells; NI = not identified; - = negative reaction;
In the reactive sera, the agglutination test seemed to be more sensitive than the immunofluorescence test: in fact, in 11 patients, the average score of GAT and LAT was higher than the GIFT score; the opposite was found only in 3 cases; agglutination and immunofluorescence tests were identical only in 2 patients.
50
100 60 40
BorzidAssali
100
NI
100
NI
100 50 70 15 0 0 0 0 0
NI NA 1 NI NI
+ = positive reaction; w = weak reaction.
In 10 patients, granulocyte cytotoxic antibodies were present. These antibodies were not strictly related with the results of the other assays: in most cases they were detected in sera giving high score results with GAT, LAT and GIFT, but in other cases GCT was positive in spite of weak reactions with those assays (cases 2 and 3).
Granulocyte Serology
Lymphocytotoxic antibodies were negative in all but 2 doubtful cases (cases 4 and 12). The neutrophil-specific antibodies reacted with various percentages of the donor panel: from 15% up to 100%. In strongly reacting sera, the percentage could be related to the alloantigen phenotype of the donor cells. In weakly reacting sera, a lower percentage of donors was recognized; this finding was possibly related with the sensitivity of the assays. All but 1(case 10) positive serum contained neutrophilspecific IgG antibodies. In 4 sera, it was possible to demonstrate the NA1 alloantigen specificity of the involved antibodies.
Discussion
A cause-effect relationship was demonstrated between the presence of neutrophil-specific antibodies and the development of symptomatic neutropenia in children as well as in adults [4,5,14-171. Our data confirmed this correlation. In all the patients investigated during the symptomatic phase of their disease, antineutrophil antibodies were detectable in the serum, the peripheral neutrophil count was low, a myeloid series maturation arrest was evident, and the prevalence of infectious episodes was higher than normal. Eleven sera from 10 patients investigated after complete symptomatic recovery were found negative. Greatly variable score reactions were found among the different donor granulocytes tested with the same patients’ sera. Such score reaction variability was slightly more evident in GAT and LAT than in GIFT. Autoantibodies detectable in autoimmune neutropenia almost invariably recognize neutrophil alloantigens [8, 18, 191; these antigens are transmitted by biallelic systems [6, 201. The difference in the reaction strength may be related with at least two possible dose-effect phenomena: (a) the single or double antigen dose related to the genetic transmission of the character and (b) the variable individual expression of the FcRIII which carries the ‘private’ specificities of the neutrophil NA system [21-231. Although some authors reported that GIFT is generally more sensitive than GAT [5,24], in this series of patients we found the opposite. In 11/16 patients, the average agglutination score was higher than the average immunofluorescence score. The functional and immunological characteristics of the antigens and antibodies involved, and the intrinsic sensitivity of the various tests may be responsible for the polymorphism of the serological reactions. The antigen-
antibody binding affinity may influence the results of the immunofluorescence assay: in fact, low-affinity antibodies may be removed from the target cells by washings [lo]. GAT and LAT gave overlapping results; GAT proved to be slightly more sensitive than LAT, probably because of the higher purity of the granulocyte suspension. On the basis of cost-benefit considerations, LAT is more indicated for screening investigations, while GAT is more indicated for investigations with clinical and therapeutic implications. The cytotoxicity assay (GCT) gives information about the in vitro complement fixation capacity of neutrophilspecific antibodies. In our series of investigations, the test was found to be positive only in 10 patients. A higher threshold for the complement-mediated antibody-dependent cytolysis and the influence of the different IgG subclasses (not investigated) are the possible reasons for its lower sensitivity. Autoantibodies responsible for autoimmune neutropenia recognize allospecific antigens [4, 7, 8,16,18, 191. This feature is poorly understood. We propose that autorecognition of so-called alloantigens is possibly triggered by neutrophil binding of immune complexes and exposition to haptenic cryptantigens of the Fc receptors, following infection: in fact, a certain seasonal spreading of idiopathic juvenile autoimmune neutropenia was observed (cold seasons: data not reported). Autorecognition may arise from the combined effect of: (a) the structural heterogeneity of human Fcy receptors; (b) the class I1 major histocompatibility complex constitution of the patients [25], and (c) some possible immaturity of the helper-suppressor idiotype network owing to the age of the patients. It is known that the involved autoantibodies are mostly directed against the NA1 alloantigen [4]. We could demonstrate this specificity in 4 patients. In some other cases, technical problems and the availability of weakly reacting antibodies prevented us from properly characterizing their specificity. In spite of their alloantigen specificity, the antibodies implicated in autoimmune neutropenia are truly autoreactive. In 8 cases (data not reported) we could perform both the ‘direct’ test and the ‘indirect’ test in the early recovery phase using stored sera and freshly collected neutrophils during the permanent recovery phase. Autoreactivity was demonstrated in all cases. Two different assays may give indirect functional information about neutrophil antibodies: the GCT and the characterization of the IgG subclasses. As discussed above, in our hands GCT proved to be less sensitive than other tests. IgG subclass characterization was not per-
51
formed. Such investigation might add useful information, but it is not suitable for a complex multiassay investigation. It should be performed separately, using known strong reacting target cells. The simultaneous performance of the agglutination test and the immunofluorescence assay is a fundamental rule in neutrophil serology, owing to their differentiated antigen-antibody recognition capacity. In autoimmune neutropenia, the application of this principle is particularly useful for the detection of antibodies with possible functional and immunological differences. In addition, it seems to be useful to repeat the investigations after the
clinical recovery. The serological follow-up demonstrated that, sometimes, complete clinical remission takes place before the antineutrophil antibodies became undetectable. This probably means that, at the last stage of the disease, a dynamic disequilibrium exists between the high bone marrow myeloid potential and the cytotoxic capacity of the residue antibody, with a neat advantage of the neutrophil function. In conclusion, the neutrophil-specific serological investigation, by a multiassay procedure, provides a contribution to the diagnosis and clinical follow-up of the patients affected by juvenile autoimmune neutropenia.
................................................................................................................................................... References 1 Salomonsen L: Granulocytopenia in children. Acta Pediatr Scand 1948;35:189-201. 2 Weetman RM, Boxer LA: Childhood neutropenia. Pediatr Clin North Am 1980;27:361375. 3 Madyastha PR, Fudenberg HH, Glassman AB, Ramananda M, Smith CL: Autoimmune neutropenia in early infancy: A review. Ann Clin Lab Sci 1982;12:356-367. 4 Lalezari P, Khorshidi M, Petrosova M: Autoimmune neutropenia of infancy. J Pediatr 1986;109:764-768. 5 Conway LT, Clay ME, Kline WE, Ramsay NKC, Krivit W, McCullough J: Natural history of primary autoimmune neutropenia in infancy. Pediatrics 1987:79:728-733. 6 Lalezari P, Radel E: Neutrophil-specific antigens: Immunology and clinical significance. Semin Hematol 1974;11:281-290. 7 McCullough J , Clay M, Press C, Kline W Clinical significance of granulocyte antigens and antibodies; in Granulocyte Serology: A Clinical and Laboratory Guide. Chicago, ASCP Press, 1988, pp 83-112. 8 Lalezari P: Neutrophil antigens: Immunology and clinical implications; in Greenwalt TJ, Jamieson GA (eds): Progress in Clinical Biological Research. New York, Liss, 1977, vol13, pp 209-225. 9 De Araujo AC, De Los Santos Fortuna E, Ionemoto HF, Jacob CMA: Neutropenia in an infant: Possible autoimmune etiology. Brazil J Med Biol Res 1989;22:967-969. 10 Verheugt GWA, Von Dem Borne AEGKr, Decary F, Engelfriet CP: The detection of granulocyte alloantibodies with a indirect immunofluorescence test. Br J Haematol 1977:36: 533-544.
11 Lalezari P, Pryce SC: Detection of neutrophil and platelet antibodies in immunologically induced neutropenia and thrombocytopenia; in Rose NR, Friedmann H (eds): Manual of Clinical Immunology, Washington, American Society of Microbiology, 1980, pp 744-749. 12 Terasaki PI, Bernoco D , Park MS, Ozturk G , Iwaki Y: Microdoplet testing for HLA-A, -B, -C, and -D antigens. Am J Clin Pathol 1978: 69:103-120. 13 Hasegawa T , Graw RG, Terasaki PI: A microgranulocyte cytotoxicity test. Transplantation 1973;15:492-498. 14 Boxer LA, Greenberg MS, Boxer GJ, Stossel TP: Autoimmune neutropenia. N Engl J Med 1975;293:748-753. 15 Harmon DC, Weitzman SA, Stossel TP: The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies. Br J Haematol 1984;58:209215. 16 Minchinton RM, Waters AH: The occurrence and significance of neutrophil antibodies. Br J Haematol 1984;56:521-528. 17 Ducos R, Madyastha PR, Warrier RP, Glassman AB, Shirley LR: Neutrophil agglutinins in idiopathic chronic neutropenia of early childhood. Am J Dis Child 1986;140:65-68. 18 Verheugt FWA, von dem Borne AEGKr, van Noord-Bochorst JC, Engelfriet CP: Autoimmune granulocytopenia: The detection of granulocyte autoantibodies with the immunofluorescence test. Br J Haematol 1978;39:339-350, 19 Sabbe LJM, Claas FHJ, Langerak J, Claus G, Smit G , Konong LWA, De Schreuder CH, van Rood JJ: Group-specific auto-immune antibodies directed to granulocytes as a cause of benign neutropenia in infants. Acta Haematol 1983;68:20-27.
20 Verheugt FWA, van dem Borne AEGKr, van Noord-Bokhorst JC, Nijenhuis LE, Engelfriet CP: ND1, a new neutrophil granulocyte antigen. Vox Sang 1978;35:13-17. 21 Verner G , von dem Borne AEGKr, Bos MJE, Tromp JF, van Plasvan Dalen CM, Visser FJ. Engelfriet CP, Tetteroo PAT Localization of the human NAl alloantigen on neutrophil-Fc gamma receptors: in Reinherz E (ed): Leukocyte Testing 11: Human Leukocyte Differentiation Antigens Detected by Monoclonal Antibodies. Berlin, Springer, 1986, pp 109-122. 22 Huizinga TW,Kleiser M, Ross D , von dem Borne AEGKr: Differences between FcR 111 of human neutrophils and human lymphocytes in relation to the NA antigen system: in Knapp W, Dorken B, Schmidt RE, Stein H, von dem Borne AEGKr (eds): Leukocyte Typing IV. Oxford, Oxford University Press, 1989. 23 Bierling P, Poulet E, Fromont P, Seror T. Bracq C, Duerari N: Neutrophil-specific antigen and gene frequencies in the French population. Transfusion 1990:30:848-849. 24 Verheugt FWA, von dem Borne AEGKr, van Noord-Bokhorst JC, van Elven EH, Engelfriet CP: Serological, immunochemical and immunocytological properties of granulocyte antibodies. Vox Sang 1978;35:294-303. 25 Bux J, Mueller-Eckhardt G , Mueller-Eckhardt C: Autoimmunization against neutrophil specific NA1 antigen is associated with HLA-DR. Hum Iminunol 1991;30:18-21.
52
Borzini/Assali
Granulocyte Serology
Piero Borzini Giorpio Assali CentrYu Trasfusionale e Laboratorio di Ematologia, Ospedale San Gerardo, Monza, Italia
Granulocyte Serology Findings in Juvenile Symptomatic ldiopathic Autoimmune Neutropenia Using a Multiassay Procedure Report on 21 Cases
................................................................................................. Abstract The serological findings on 21 children with idiopathic autoimmune neutropenia are reported. A multiassay procedure was adopted including agglutination, immunofluorescence and cytotoxicity tests. Beside a cause-effect correlation between granulocyte antibodies and clinical course, a serologic polymorphism was found. As the sensitivity of each assay seemed to be related to the characteristics of the involved antigens and antibodies, as well as to the intrinsic sensitivity of the tests, the performance of a multiassay procedure appears to be advisable for the diagnosis and the follow-up of autoimmune neutropenia.
.....................
Introduction
Juvenile idiopathic autoimmune neutropenia is a welldocumented, although uncommon, children's disease [1-4]. Children are affected mainly during the first 2 years of life [ 5 ] ;the clinical course is generally benign with spontaneous resolution within 6-24 months from onset, without sequelae [5-71. The neutropenia is very often associated with the presence of auto- and alloreactive neutrophil-specific antibodies; most of these antibodies recognize polymorphic alloantigens and the immunopathogenetic origin of immunization is still unclear [4,8, 91. We report the serologic findings of 21 children with symptomatic idiopathic neutropenia. Findings were quite uniform and in good agreement with the clinical condi-
Received: Jan. 18,1991 Revised manuscript received: Nov. 25. 1991 Accepted: Nov. 27, 1991
tion. Some differences in the reactivity of the serum antibodies with the target cells were consistent with an immunologic polymorphism of the disease.
Materials and Methods Patients Twenty-one patients were investigated. The patients' blood samples were referred to our laboratory from the pediatric divisions of eight different hospitals. In all cases the peripheral blood smears were observed, while wecouldexamine the bone marrow specimens in only one third of the patients (those referred from our hospital). At the time of the first observation, 16 patients were symptomatic while 5 were free of symptoms and probably entering the recovery phase.
Dr. Piero Borzini Centro Trasfusionalee Lahoratorio di Ematologia Ospedale San Gerardo Via Snlferino. I6 20052 Monza (Italy)
0 1992 S. Karger AG. Basel 0042-90071921Oh314W8 $2.7510
Serologic Investigations The following investigations were performed: the indirect granulocyte suspensionimmunofluorescence test (GIFT), the leukoagglutination test (LAT), the granulocyte agglutination test (GAT), the granulocyte cytotoxicity test (GCT), and the lymphocytotoxicity test (LCT). The target cells were obtained from a panel of at least twelve random (0 blood group) blood donors. The NA1 specificity was determined using a panel of 6 donors with known NA1 phenotype. Target Cells Leukocytes to be used as target cells for the LAT were isolated from blood collected in NazEDTA (10 mM final concentration). One milliliterofdextran6% (w/v) wasaddedto5 mlofwholeanticoagulated blood and the volume was brought to 10 ml with 0.15 Mphosphatebuffered saline (PBS) pH 7.2. The tubes were incubated for 30 min at 37°C. The supernatant was then collected and centrifuged. The cells obtained were washed three times with PBS containing 8 mM EDTA (PBS-EDTA). After the last wash the contaminating erythrocytes were submitted to hypotonic lysis with a short incubation (40 s) with 33 mM NaCI. The leukocytes were washed three times with PBSEDTA containing 0.3% bovine serum albumin (PBS-EDTA-BSA). The granulocytes to be used for the granulocyte agglutination test (GAT) andfortheGIFTwereobtained asfollows. Theleukocyte-rich suspension was obtained as described above, and the lymphocytes were removed by gradient sedimentation (d = 1.077).The neutrophils were collected from the bottom of the tubes. After washings with PBS-EDTA-BSA, they were resuspended in the same buffer and used to perform GAT. A portion of the granulocyte suspension was fixed with 1%paraformaldehyde solution, pH 7.2, for5 min at 22°C. After three washings with PBS-EDTA-BSA, they were used to perform GIFT. Lymphocytes and granulocytes for the cytotoxicity assays (LCT and GCT) were obtained by dextran sedimentation followed by gradient sedimentation, as described above, using blood collected in heparin (1 unit/ml) and using RPMI-1640 with Hepes, containing20Y0 fetal calf serum (FCS), as working buffer (RPMI-FCS). Assays GIFT GIFT was performed on paraformaldehyde-fixed granulocytes [lo]. The fixed washed cells were incubated with patients' sera for30 min at 37"Cfor IgG detection and at room temperature for IgM detection. Class-specificFITCF(ab')2antihuman IgG and antihuman IgM were used for isotype characterization. GATand LAT GAT and LAT were performed using unfixed cells [lo, 111,incubating 50 pl of cell suspension (at 3-5 x lO'/ml) with 100 111 of serum, for 2 h. LCT and GC?: LCT and GCT were performed in 60 microwell tissue-typing trays [12,13]. The cells were resuspended in RPMI-FCS and brought to 4-6 x 106/ml.LCT was performed using standard NIH conditions, while, for GCT, the complement-mediated lysis was prolonged for 3 h. Scoring The positive reactions were scored as follows: agglutination and immunofluorescence tests: from t w to t t (subjective evaluation); cytotoxicity asssays: from 2 to 8 (according to the percentage of cell death).
+
ResuIts
The mean age of the 21 patients was 16.4 months (range 9-40, median 14). Sixteen patients entered the study in a symptomatic phase of the disease and 5 were subjected to a unique serological investigation some months after symptomatic recovery. All patients considered together had a median of 8 infections per year during the symptomatic course of their disease. A great variety of muco-epidermal infections sustained by a variety of gram-positive and gram-negative bacteria were reported. Curative antibiotic treatment and highdose intravenous gammaglobulins were administered during the acute phase of the disease. Chronic phases were variously treated: in most cases, prednisone (1 mgl kg/day) and prophylactic antibiotics were administered when the neutrophil count was below 500/p1. No patient received leukocyte transfusions. The leukocyte count was in a wide range (1,100-18,000) while the neutrophil count was markedly low in the symptomatic patients (range 0-650/p1; mean 400; median 500) in comparison with the asymptomatic patients (range 1,000-2,100; mean 1,400; median 1,200). The differential leukocyte count of the peripheral blood was characterized by the presence of a discrete percentage (5-20%) of mononuclear cells deriving from the myeloid series (myelocytes and degranulated metamyelocytes). In 7 symptomatic cases, the observation of bone marrow specimens revealed myeloid hyperplasia, the absence of mature and hypersegmented neutrophil, and an arrest of maturation at myelocyte or metamyelocyte stage. Table 1represents the serological findings of the 39 sera from the 21 patients studied. No antibody was found in 5 patients (23%). These patients are listed at the end of the table: all of them were free of symptoms and their neutrophil count was within the normal values or borderline. Sixteen patients (77%) were serologically positive: 6 out 34 sera from these patients were negative at the last observation. These patients (cases 4-6, 10 and 12) were considered clinically cured at the time of the last observation. Two other patients (cases 3 and 8) were clinically cured at the time of the last observation: their sera were still reactive in the agglutination assay, while the peripheral neutrophil count was within the normal range. One interesting technical finding was the evidence of variable scores of the positive reactions obtained with the different donor's target granulocytes tested with the same sera. This finding will be discussed later.
49
Table 1. Age, observation date, neutrophil count per microliter and serological findings in 21 children affected with idiopathic juvenile autoimmune neutropenia
Case
Age months
Date
PMN
GAT
13 21 15
2/88 11/88 1/88 1/89 2/88 4/88 6/88 12/88 3/88 4/88 5/88 7/88 1/89 4/88 5/88 6/88 12/88 6/88 7/88 7/89 11/89 12/89 4/89 4/89 2/90 10190 11/90 12/89 1/90 2/90 2/90 3/90 12/90 12/90 3/88 5/88 1/89 7/89 1/90
150 500 500 600 50 50 550 950 650 600 1100 1750 1650 650 750 900 1200 500 500 450 650 650 0 350 2000 300 800 400 850 1000 80 550 6.50 650 1200 1500 1000 2100 1200
+w/+
LAT
GIFT
+w/++
+W/+t
GCT
LCT
Percent Ig class of panel
Specificity
-
1 2 3
4
5
6
7 8
16
12
13
14 24
9 10
12 15
11
15
12 13 14 15 16 17 18 19 20 21
20 12 39 10 10 9 40 17 9 9
++
+W
-
CW
+W
+W
+W
+W
-
+ + / + + t++I+++ +I+++ +I+ t
+
+w/++ +w/+t
50
NI NI NI NA 1
+W
+W
-
-
-
+I+
+
-
-
+
100
NI
25
NI
20
NI
50
NAI
50 60
NA 1 NI
+W
-
-
+
-
+W +W
-
+ + +/+ + + +W +++
+W +W
t +W +W
-
+I+++ -
+++ +W
+/++
+
+W
-
++ +W +
+W
-
-
-
+++ +
+++
+W
+W
+
+W
-
-
-
-
-
-
-
-
-
-
PMN = Neutrophil polymorphonuclear cells; NI = not identified; - = negative reaction;
In the reactive sera, the agglutination test seemed to be more sensitive than the immunofluorescence test: in fact, in 11 patients, the average score of GAT and LAT was higher than the GIFT score; the opposite was found only in 3 cases; agglutination and immunofluorescence tests were identical only in 2 patients.
50
100 60 40
BorzidAssali
100
NI
100
NI
100 50 70 15 0 0 0 0 0
NI NA 1 NI NI
+ = positive reaction; w = weak reaction.
In 10 patients, granulocyte cytotoxic antibodies were present. These antibodies were not strictly related with the results of the other assays: in most cases they were detected in sera giving high score results with GAT, LAT and GIFT, but in other cases GCT was positive in spite of weak reactions with those assays (cases 2 and 3).
Granulocyte Serology
Lymphocytotoxic antibodies were negative in all but 2 doubtful cases (cases 4 and 12). The neutrophil-specific antibodies reacted with various percentages of the donor panel: from 15% up to 100%. In strongly reacting sera, the percentage could be related to the alloantigen phenotype of the donor cells. In weakly reacting sera, a lower percentage of donors was recognized; this finding was possibly related with the sensitivity of the assays. All but 1(case 10) positive serum contained neutrophilspecific IgG antibodies. In 4 sera, it was possible to demonstrate the NA1 alloantigen specificity of the involved antibodies.
Discussion
A cause-effect relationship was demonstrated between the presence of neutrophil-specific antibodies and the development of symptomatic neutropenia in children as well as in adults [4,5,14-171. Our data confirmed this correlation. In all the patients investigated during the symptomatic phase of their disease, antineutrophil antibodies were detectable in the serum, the peripheral neutrophil count was low, a myeloid series maturation arrest was evident, and the prevalence of infectious episodes was higher than normal. Eleven sera from 10 patients investigated after complete symptomatic recovery were found negative. Greatly variable score reactions were found among the different donor granulocytes tested with the same patients’ sera. Such score reaction variability was slightly more evident in GAT and LAT than in GIFT. Autoantibodies detectable in autoimmune neutropenia almost invariably recognize neutrophil alloantigens [8, 18, 191; these antigens are transmitted by biallelic systems [6, 201. The difference in the reaction strength may be related with at least two possible dose-effect phenomena: (a) the single or double antigen dose related to the genetic transmission of the character and (b) the variable individual expression of the FcRIII which carries the ‘private’ specificities of the neutrophil NA system [21-231. Although some authors reported that GIFT is generally more sensitive than GAT [5,24], in this series of patients we found the opposite. In 11/16 patients, the average agglutination score was higher than the average immunofluorescence score. The functional and immunological characteristics of the antigens and antibodies involved, and the intrinsic sensitivity of the various tests may be responsible for the polymorphism of the serological reactions. The antigen-
antibody binding affinity may influence the results of the immunofluorescence assay: in fact, low-affinity antibodies may be removed from the target cells by washings [lo]. GAT and LAT gave overlapping results; GAT proved to be slightly more sensitive than LAT, probably because of the higher purity of the granulocyte suspension. On the basis of cost-benefit considerations, LAT is more indicated for screening investigations, while GAT is more indicated for investigations with clinical and therapeutic implications. The cytotoxicity assay (GCT) gives information about the in vitro complement fixation capacity of neutrophilspecific antibodies. In our series of investigations, the test was found to be positive only in 10 patients. A higher threshold for the complement-mediated antibody-dependent cytolysis and the influence of the different IgG subclasses (not investigated) are the possible reasons for its lower sensitivity. Autoantibodies responsible for autoimmune neutropenia recognize allospecific antigens [4, 7, 8,16,18, 191. This feature is poorly understood. We propose that autorecognition of so-called alloantigens is possibly triggered by neutrophil binding of immune complexes and exposition to haptenic cryptantigens of the Fc receptors, following infection: in fact, a certain seasonal spreading of idiopathic juvenile autoimmune neutropenia was observed (cold seasons: data not reported). Autorecognition may arise from the combined effect of: (a) the structural heterogeneity of human Fcy receptors; (b) the class I1 major histocompatibility complex constitution of the patients [25], and (c) some possible immaturity of the helper-suppressor idiotype network owing to the age of the patients. It is known that the involved autoantibodies are mostly directed against the NA1 alloantigen [4]. We could demonstrate this specificity in 4 patients. In some other cases, technical problems and the availability of weakly reacting antibodies prevented us from properly characterizing their specificity. In spite of their alloantigen specificity, the antibodies implicated in autoimmune neutropenia are truly autoreactive. In 8 cases (data not reported) we could perform both the ‘direct’ test and the ‘indirect’ test in the early recovery phase using stored sera and freshly collected neutrophils during the permanent recovery phase. Autoreactivity was demonstrated in all cases. Two different assays may give indirect functional information about neutrophil antibodies: the GCT and the characterization of the IgG subclasses. As discussed above, in our hands GCT proved to be less sensitive than other tests. IgG subclass characterization was not per-
51
formed. Such investigation might add useful information, but it is not suitable for a complex multiassay investigation. It should be performed separately, using known strong reacting target cells. The simultaneous performance of the agglutination test and the immunofluorescence assay is a fundamental rule in neutrophil serology, owing to their differentiated antigen-antibody recognition capacity. In autoimmune neutropenia, the application of this principle is particularly useful for the detection of antibodies with possible functional and immunological differences. In addition, it seems to be useful to repeat the investigations after the
clinical recovery. The serological follow-up demonstrated that, sometimes, complete clinical remission takes place before the antineutrophil antibodies became undetectable. This probably means that, at the last stage of the disease, a dynamic disequilibrium exists between the high bone marrow myeloid potential and the cytotoxic capacity of the residue antibody, with a neat advantage of the neutrophil function. In conclusion, the neutrophil-specific serological investigation, by a multiassay procedure, provides a contribution to the diagnosis and clinical follow-up of the patients affected by juvenile autoimmune neutropenia.
................................................................................................................................................... References 1 Salomonsen L: Granulocytopenia in children. Acta Pediatr Scand 1948;35:189-201. 2 Weetman RM, Boxer LA: Childhood neutropenia. Pediatr Clin North Am 1980;27:361375. 3 Madyastha PR, Fudenberg HH, Glassman AB, Ramananda M, Smith CL: Autoimmune neutropenia in early infancy: A review. Ann Clin Lab Sci 1982;12:356-367. 4 Lalezari P, Khorshidi M, Petrosova M: Autoimmune neutropenia of infancy. J Pediatr 1986;109:764-768. 5 Conway LT, Clay ME, Kline WE, Ramsay NKC, Krivit W, McCullough J: Natural history of primary autoimmune neutropenia in infancy. Pediatrics 1987:79:728-733. 6 Lalezari P, Radel E: Neutrophil-specific antigens: Immunology and clinical significance. Semin Hematol 1974;11:281-290. 7 McCullough J , Clay M, Press C, Kline W Clinical significance of granulocyte antigens and antibodies; in Granulocyte Serology: A Clinical and Laboratory Guide. Chicago, ASCP Press, 1988, pp 83-112. 8 Lalezari P: Neutrophil antigens: Immunology and clinical implications; in Greenwalt TJ, Jamieson GA (eds): Progress in Clinical Biological Research. New York, Liss, 1977, vol13, pp 209-225. 9 De Araujo AC, De Los Santos Fortuna E, Ionemoto HF, Jacob CMA: Neutropenia in an infant: Possible autoimmune etiology. Brazil J Med Biol Res 1989;22:967-969. 10 Verheugt GWA, Von Dem Borne AEGKr, Decary F, Engelfriet CP: The detection of granulocyte alloantibodies with a indirect immunofluorescence test. Br J Haematol 1977:36: 533-544.
11 Lalezari P, Pryce SC: Detection of neutrophil and platelet antibodies in immunologically induced neutropenia and thrombocytopenia; in Rose NR, Friedmann H (eds): Manual of Clinical Immunology, Washington, American Society of Microbiology, 1980, pp 744-749. 12 Terasaki PI, Bernoco D , Park MS, Ozturk G , Iwaki Y: Microdoplet testing for HLA-A, -B, -C, and -D antigens. Am J Clin Pathol 1978: 69:103-120. 13 Hasegawa T , Graw RG, Terasaki PI: A microgranulocyte cytotoxicity test. Transplantation 1973;15:492-498. 14 Boxer LA, Greenberg MS, Boxer GJ, Stossel TP: Autoimmune neutropenia. N Engl J Med 1975;293:748-753. 15 Harmon DC, Weitzman SA, Stossel TP: The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies. Br J Haematol 1984;58:209215. 16 Minchinton RM, Waters AH: The occurrence and significance of neutrophil antibodies. Br J Haematol 1984;56:521-528. 17 Ducos R, Madyastha PR, Warrier RP, Glassman AB, Shirley LR: Neutrophil agglutinins in idiopathic chronic neutropenia of early childhood. Am J Dis Child 1986;140:65-68. 18 Verheugt FWA, von dem Borne AEGKr, van Noord-Bochorst JC, Engelfriet CP: Autoimmune granulocytopenia: The detection of granulocyte autoantibodies with the immunofluorescence test. Br J Haematol 1978;39:339-350, 19 Sabbe LJM, Claas FHJ, Langerak J, Claus G, Smit G , Konong LWA, De Schreuder CH, van Rood JJ: Group-specific auto-immune antibodies directed to granulocytes as a cause of benign neutropenia in infants. Acta Haematol 1983;68:20-27.
20 Verheugt FWA, van dem Borne AEGKr, van Noord-Bokhorst JC, Nijenhuis LE, Engelfriet CP: ND1, a new neutrophil granulocyte antigen. Vox Sang 1978;35:13-17. 21 Verner G , von dem Borne AEGKr, Bos MJE, Tromp JF, van Plasvan Dalen CM, Visser FJ. Engelfriet CP, Tetteroo PAT Localization of the human NAl alloantigen on neutrophil-Fc gamma receptors: in Reinherz E (ed): Leukocyte Testing 11: Human Leukocyte Differentiation Antigens Detected by Monoclonal Antibodies. Berlin, Springer, 1986, pp 109-122. 22 Huizinga TW,Kleiser M, Ross D , von dem Borne AEGKr: Differences between FcR 111 of human neutrophils and human lymphocytes in relation to the NA antigen system: in Knapp W, Dorken B, Schmidt RE, Stein H, von dem Borne AEGKr (eds): Leukocyte Typing IV. Oxford, Oxford University Press, 1989. 23 Bierling P, Poulet E, Fromont P, Seror T. Bracq C, Duerari N: Neutrophil-specific antigen and gene frequencies in the French population. Transfusion 1990:30:848-849. 24 Verheugt FWA, von dem Borne AEGKr, van Noord-Bokhorst JC, van Elven EH, Engelfriet CP: Serological, immunochemical and immunocytological properties of granulocyte antibodies. Vox Sang 1978;35:294-303. 25 Bux J, Mueller-Eckhardt G , Mueller-Eckhardt C: Autoimmunization against neutrophil specific NA1 antigen is associated with HLA-DR. Hum Iminunol 1991;30:18-21.
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Borzini/Assali
Granulocyte Serology
