Journal of Infection (1991) zz, 59-69

CASE REPORT H u m o r a l i m m u n e r e s p o n s e to p n e u m o c o c c a l a n t i g e n 23-F in an asplenic patient with r e c u r r e n t f u l m i n a n t pneumococcaemia W. Z i m m e r l i , * ~ A. S c h a f f n e r , t C. S c h e i d e g g e r , * R . Scherz~ and P . J. Sp~ith~

* Departments of Internal Medicine and Research, University Hospital, Basel, t Department of Internal Medicine, University Hospital, Ziirich and ~ Central Laboratory, Blood Transfusion Service, SRC, Bern, Switzerland Accepted for publication 3 June 199 °

Summary Host defence mechanisms were analysed in a patient with three episodes of fulminant pneumococcaemia and one episode of bacteraemic epiglottitis with Haemophilus influenzae type b. The first episode took place I i years after splenectomy for blunt abdominal trauma. Investigations revealed several host defence mechanisms to be impaired. In addition to the patient's asplenia, an inherited C2-deficiency was noted. Assessment of IgG subclasses repeatedly revealed markedly low IgG4 concentrations. These were not due to an increased turnover of IgG4, as could be shown following infusion of intravenous IgG. In addition, IgG2 concentrations were low in the patient who lacked G2M(23). Opsonic mediating antibodies against type 23-F pneumococci were in the range of those of non-immune volunteers 6 months after vaccination with a 23-valent pneumococcal vaccine. These antibodies did not increase after a septic episode with 23-F capsular-type pneumococci. Neutrophil function was apparently normal.

Introduction Several conditions in h u m a n beings are k n o w n to predispose to infections due to encapsulated bacteria. T h e risk of fulminant p n e u m o c o c c a e m i a is especially increased in splenectomised patients b u t to a varying degree according to age and underlying disease. 1 T h e basis of this association is not entirely understood. It is agreed that encapsulated organisms are mainly eliminated b y macrophages of the liver and spleen. In the i m m u n e host, the elimination of opsonised organisms p r e d o m i n a n t l y and rapidly takes place in the liver p r o v i d e d that the classical p a t h w a y of c o m p l e m e n t is activated. In noni m m u n e persons a m u c h slower elimination p r e d o m i n a n t l y b y the spleen is t h o u g h t to occurfl T h e role of c o m p l e m e n t in the rapid and effective elimination of such organisms is u n d e r s c o r e d b y the association of certain Address correspondence to: Dr W. Zimmerli, Departments of Internal Medicine and Research, University Hospital, CH-4o3I Basel, Switzerland.

oi63-4453/9I/OiOO59+ II $03.00/0

© I99I The British Society for the Study of Infection

60

W. Z I M M E R L I

ET AL.

complement deficiencies with severe recurrent infections by encapsulated bacteria. ~ We analysed the humoral i m m u n i t y and phagocytic function in a patient with three episodes of fulminant pneumococcaemia and one episode of bacteraemic epiglottitis caused by Haemophilus influenzae type b I I years after splenectomy for trauma and found several concomitant abnormalities of the humoral immunity. Methods Detection o f p n e u m o c o c c a l polysaccharide antigen in s e r u m by ELISA

Serum was diluted I in 5 in phosphate-buffered saline before heat inactivation (Ioo °C for Io min) and centrifugation at 3ooo g for I5 min in order to remove denatured proteins. Microtitre plates were coated with I g G from rabbits i m m u n i s e d with pools (seven to eight serotypes each) of formalin-fixed pneumococci. Five individual pools were used to immunise rabbits. T h e antibodies obtained from one pool of antigens did not show cross-reactivity with the other four pools of antigens. T h e antibodies alone served as the solid phase for the capture of antigen from patient's serum while peroxidaseconjugated antibodies were used for detecting captured antigens. T h e system included solid-phase antibodies raised against the 39 most commonly encountered serotypes of pneumococci in Switzerland. It uniformly detected antigen at concentrations of o . I - i n g / m l and did not show any detectable cross-reaction with constituents of heated body fluids or suspensions of other bacterial species. Serotyping of pneumococcal isolates or of antigen in serum was done by the Quellung reaction or the solid phase E L I S A by means of pooled h u m a n sera for initial screening. Commercially available specific antisera (Statens Serum Institute, Copenhagen, Denmark) directed against each of the 39 serotypes served for the detailed typing. Assay for opsonisation o f bacteria by s e r u m

T h e pneumococcal opsonic activity of serum was assessed by chemiluminescence induced by bacteria in h u m a n neutrophil granulocytes. A luminol amplified system was used. ~ T h e staphylococcal opsonic activity of serum was assessed in a phagocytic-bactericidal assay. ~ Assays for neutrophil functions

Neutrophil function was determined with Percoll-purified granulocytes. 6 Chemotaxis was measured by means of a micropore filter technique in a 48microwell chamber while superoxide production was measured as superoxide dismutase-inhibitable reduction of cytochrome C in a continuous assay, and bactericidal activity in a phagocytic-bactericidal assay with Staphyloccus aureus Wood 46 as test strain. 5'6 I m m u n o c h e m i c a l analyses

Concentrations of all components of complement (except S-protein and factor D) and of Ig isotypes were measured by radial immunodiffusion or nephelometry before and after administration of intravenous 7S I g G (Sandoglobulin ®, o'4 g / k g body weight). Monoclonal antibodies (Unipath, Bedford, U.K.) or commercially available monospecific antisera were used.

Recurrent fulminant pneumococcaemia

6I

Total haemolytic complement (CH5o) and haemolytic activity of the alternative pathway of complement (APH5o) were assessed by means of micro-methods with optimally sensitized sheep red blood cells or rabbit erythrocytes in presence of 2 mmol/1 Mg 2+ and 8 mmol/1 E G T A . Functional C2 was assessed according to Joise]L et al., v and functional factor D according to Harrison and Lachmann. s G2M(23) phenotype was assessed in serum by haemagglutination inhibition assay in V-shaped microtitre plates. Highly purified myeloma IgG2, G2M(23)-positive, was used as the coat for ORhh u m a n erythrocytes (coating according to the m e t h o d by Gold and F u n d e n b e r g ) 2 Anti-G2M(23) antiserum was obtained from Janssen (Beeze, Belgium, Code 24.oo3.44). C i q - b i n d i n g activity of serum samples was measured as described. 1° Case r e p o r t

A Caucasian male patient was splenectomised following blunt abdominal trauma at I3 years of age. A first episode of fulminant pneumococcaemia took place I I years after the splenectomy. It started abruptly with headache, 4o'3 °C fever and chills. His physician gave h i m a dose of 750 m g amoxicillin a few hours after the onset of the symptoms. Within I2 h, he was admitted to hospital in shock (systolic blood pressure 60 m m H g ) , afebrile, cyanotic, icteric and complained of generalised pain. Laboratory tests indicated 3I x IOg/1 leucocytes (6o~/o bands), renal failure (creatinine 47o#mol/1), hepatitis ( S G O T I 6 4 U / 1 ) and disseminated intravascular coagulation (fibrinogen I'25 g/t, factors II, V, V I I at 46, 2.4 and 3z % of the control, respectively, and fibrin degradation products > 40 #g/ml). An infectious focus was not found. Blood cultures remained sterile but pneumococcal antigen could be detected in serum on the day of admission to hospital and 2 days later. These samples of serum reacted with I g G from rabbits i m m u n i s e d by a pool of pneumococcal antigens (gN, 9V, IoF, I IB, I7F, 23F). With low-dose heparin, high-dose penicillin and vasopressors the patient rapidly recovered. T h r e e m o n t h s later, he was again admitted to hospital with the same symptoms. Streptococcus pneumoniae grew in four cultures of blood drawn before starting treatment with intravenous penicillin. This strain reacted with O m n i s e r u m ® but not with our five pools of diagnostic sera, which are directed against the 39 most c o m m o n Swiss isolates (including all vaccine strains of the 23-valent pneumococcal vaccine). Therefore, it was obviously a strain other than the one of the first episode. T h e patient recovered without problems. Six months later he was i m m u n i s e d with a 23-valent pneumococcal vaccine (Pneumovax-23 ~) without adverse reaction. T h e fbllowing m o n t h , the patient developed a bacteraemic epiglottitis with Haemophilus influenzae type b from which he recovered rapidly with amoxicillin/clavulanic acid. T h e third episode of fulminant pneumococcaemia arose 6 m o n t h s after pneumococcal vaccination. Despite admission to hospital within 2 h after the first symptoms the patient developed renal failure (creatinine z98 #mol/1), hepatitis ( S G O T 97 U / l ) and an acute psychosis. As a result of shock and respiratory distress, he required 2 days of adrenergic support and 3 days of assisted ventilation. Circulating antigen which was detected during I7 days and blood cultures revealed a pneumococcus of serotype 23-F. T h e patient recovered with large

62

w. ZIMMERLI E T AL. T a b l e I Opsonic a c t i v i t y measured in a phagocytic-bactericidal assay w i t h S t a p h y l o c c u s a u r e u s W o o d 46* No. I 2

3

Source of opsonin IO % NHS IO~o patient's serum

% Killing mean-I- S.D. (n) 77 + 8 % (5) I5---23~o (4)

5

IO % hypogammaglobulinaemic serum t Io % heat-inactivated (560/3 ° min) NHS 3+ 4

6

2+ 3

79 + 7 % (5)

7

2+ 4

o % (2)

4

o + o % (3) o + o % (5) 87 % (I)

Comment/ conclusion Normal value 2 vs. r : P < o'oo5:patient's serum defective in its opsonic activity Complement source for 5 and 6 Immunoglobulin source for 5 and 7 Opsonisation requires antibodies and complement Anti-staphylococcal antibodies not deficient in patient's serum Complement deficiency in patient's serum

NHS: pooled normal human serum. * Serum (opsonic source) was incubated with 3 × IO6 CFU S. aureus Wood 46 and 5 ×IOS PMN (normal volunteer) at 37 °C in a shaking waterbath (I2o rev/min). The killing rate was determined with quantitative cultures from samples drawn at time zero and 35 min : IOO %

c r u 35 min × IO0.

t Hypogamrnaglobulinaemic serum with I'4 g/1 IgG and no measurable IgA and IgM was kindly provided by Dr J. P. Despont (Division of Immunology, University Hospital, Geneva, Switzerland).

doses o f penicillin. B e c a u s e o f t h e r e c o r d e d failure o f v a c c i n a t i o n , oral p r o p h y l a x i s w i t h 2 × 75o m g amoxicillin p e r day was started. W i t h this m e d i c a t i o n t h e p a t i e n t d i d n o t h a v e f u r t h e r severe i n f e c t i o n s d u r i n g t h e 34 m o n t h s o f s u r v e i l l a n c e a f t e r t h e last episode. Results

In view of these unusually frequent and severe infections we tested the p h a g o c y t i c a n d h u m o r a l i m m u n i t y o f the p a t i e n t . V a r i o u s g r a n u l o c t y e f u n c t i o n s s u c h as c h e m o t a x i s to f o r m y l p e p t i d e (IO-7M F M L P : 89___7%; n o r m a l v a l u e 6 o - 9 o % ) , s u p e r o x i d e p r o d u c t i o n in r e s p o n s e to IO -6 M F M L P (2"1-[-O"15 n m o l / m i n / I o 6 g r a n u l o c y t e s ; n o r m a l v a l u e 2 " 4 _ o ' 3 5 ) , a n d b a c tericidal a c t i v i t y against o p s o n i s e d S. aureus W o o d 46 (76 % killing o f t h e i n o c u l u m ; n o r m a l v a l u e 7 o - 9 o % ) w e r e all n o r m a l . I n o r d e r to test t h e role o f c o m p l e m e n t a n d i m m u n o g l o b u l i n s as o p s o n i n s , we e x a m i n e d t h e p a t i e n t ' s s e r u m in a p h a g o c y t i c - b a c t e r i c i d a l assay u s i n g S. aureus W o o d 46. O p t i m a l o p s o n i s a t i o n o f this strain was f o u n d to d e p e n d o n c o m p l e m e n t a n d i m m u n o g l o b u l i n s f r o m n o r m a l p o o l e d h u m a n s e r u m , as s h o w n in T a b l e I. T h e o p s o n i c activity o f t h e p a t i e n t ' s s e r u m was significantly reduced and could be reconstituted by complement but not by immuno-

Table II Complement analyses* Patient (25 years)

Sister (26 years)

Mother (5I years)

Normal range

Immune complexes C i q - b i n d i n g activity

2'I -}- I'8

I8

O

K 5 ~o

Immunological measurements C2 (RID) o o 53 67-I27 % All other complement proteins within the normal ranges. Functional determinations Cz < 5 < 5 58 62-I34 % Factor D

IO7 _+7"5

IO6

III

CH5ot APH 5o:~

I8'3 ±5"I 34'3 +- I'2

I5'5 33

299 36

65 -155 %

3oo-5 Io U / m l 25-55 U / m l

* Samples drawn at clinically silent periods. The results are given as mean_+ s.D. from analyses of three different samples of serum drawn at intervals > I month. Results without S.D. were means from determinations of two different samples or a single determination (mother). o, Not detectable. t I U of total haemolytic activity corresponds to the volume of serum which lyses 5o % of 3 × IO7 optimally sensibilised sheep red blood cells in 45 min at 37 °C in a total volume ofo'5 ml. I U of alternative pathway haemolytic activity corresponds to the volume of serum which lyses 50 % of 5 x io 7 rabbit erythrocytes at physiological ionic strength in the presence of 2 mmol/1 Mg 2÷ and 8 mmol/1 E G T A in 45 min at 37 °C in the total volume of 0"5 ml.

globulins (Table I). This p r o m p t e d us to investigate the patient's complement system. Results are summarised in Table II. We found a complete, selective immunological and functional deficiency of C2. T h e CH5o was < 5 ~o of the mean value of normal persons. All other complement proteins except Sprotein were tested and found normal. T h e C2-deficiency proved to be inherited, the patient's sister also being homozygously deficient in C2 while his m o t h e r was heterozygously deficient. In contrast to the patient, his sister never suffered from severe infections. She had increased C I q - b i n d i n g activity in her serum without clinical evidence of a connective tissue disease. Concentrations of IgG, IgA, I g M and IgE in the serum were in the normal range for all members of the family (data not shown). Results of the I g G subclass quantitation are summarised in Table III. IgG2 was in the lower range of the normal population in all three family members. An analysis of the possible background of the borderline IgG2 concentrations revealed G g M (23) phenotype to be absent in all family members. T h e concentration of IgG4 was very low in the patient and his C2-deficient sister but not in his m o t h e r with the heterozygous C2-deficiency. In vivo decay of the concentration of IgG4 in the patient was consistent with a normal IgG4 turnover, as was.recorded after a single infusion of 7S I g G (Table IV). Figure I shows that the concentration of opsonic antibodies against serotype 23-F pneumococci was similar to those from five n o n - i m m u n e volunteers, not only 6 m o n t h s after pneumococcal vaccination, but also 3 weeks after sepsis with type 23-F pneumococcus. T h e positive control (rabbit h y p e r i m m u n e serum to serotype 23-F; no h u m a n h y p e r i m m u n e serum available) showed

64

W. ZIMMERLI E T A L .

g~8~ O0

•

.

°

I

¢q

~

i

0

°

~&66

~ 6 6

0 ~ 0 0

"~o

oO 1¢~

8o~-

,h&66

oo

~ 9 9

0 0 0 0

+1+1+1+1

[.-,

-7-1

Recurrent fulminant pneumococcaemia

65

T a b l e IV In vivo decay of IgG subclass concentrations in a splenectomised and C2-deficient patient following infusion of a single dose of intravenous IgG (0"4 g/kg b.w.)

Before 30 min after I day after 2 days after 4 days after 5 days after 6 days after 7 days after 14 days after 31 days after

IgGI 3"55-II'Z5 (g/l)

IgG2 1'3o-5"9o (g/i)

IgG3 o"15-1"o5 (g/l)

IgG4 o'Io-I'IO (g/l)

9"35 lO'12 11"14 9"5 9"93 9"74 9"o2 9'44 8'84 8'20

1"39 2"61 2"73 2"05 2'48 2"19 2'11 2'05 2'o6 1"71

0'51 0'75 o'73 o'66 o'51 o'51 o'49 0"48 o'48 0"48

0'03 o"14 o'16 o'II o'Io o'o9 o'o9 0'08 o'o9 0-08

L03

°/°" v

E

10 a

oo

0.0-0-0-0-0-0-0-0

!

O / 9-A'A-A-A-A-A-A-A_A_A_A_A

ID

g

i

/

/

0~0"

I0'

/A l/---A-A;O=*'t=I't-*:¢-A-A-A ,7./- ze -e-e-e IIA

_ ~e

Io

15

20

Min

Fig. I. Chemiluminescence by human neutrophils induced by pneumococcal serotype 23-F in the presence of serum from the patient 6 months after pneumococcal vaccine including serotype 23-F (z~) and 21 days after bacteraemia with serotype 23-F (A), of control serum from non-immune volunteers (4~), and of rabbit hyperimmune serum to serotype 23-F (O). All sera were heat inactivated (56 °C/45 rain). Each point depicts the mean value from triplicate wells. JIN 22

66

W. Z I M M E R L I

ET AL.

that complement was not needed for efficient opsonisation since the test was performed with heat-inactivated serum. Discussion

Anatomical and functional asplenia predisposes to severe infections with encapsulated bacteria. 1' 11-15This risk is lower in patients who have undergone splenectomy for trauma compared to those for whom the spleen has been removed for thalassaemia or malignant blood diseases. L 13 Most cases of postsplenectomy sepsis arise within 2 years after surgery. 16-1s T h e increased risk for septacaemia is thought to be due to impaired clearance of intravascular unopsonised particles, low synthesis of specific antibodies, and possible abnormalities of the alternative complement pathway. 1'19 T h e latter defects are the subject of controversy. Several authors have shown that complement activity is normal in splenectomised patients. 2°'2~ In addition, the antibody response to pneumococcal vaccine has been shown to be similar in normal and splenectomised persons, except in those with underlying malignant disease. 22-25 According to a serotype surveillance study from the U.S.A., the estimated efficacy of the pneumococcal vaccine was 85 % in patients with splenic dysfunction, excluding those with sickle-cell disease. 26 Thus the three episodes of fulminant pneumococcaemia within I4 months were extremely unusual and cannot be explained by asplenia alone. It was therefore likely that this patient had multiple host-defence defects which additively increased the risk for infection with encapsulated bacteria. T h e complement analysis revealed an isolated complete C2-deficiency. T h e inheritance of this autosomal codominantly transmitted defect could be proved by family study, with a homozygous deficiency in his sister and a heterozygous one in his mother. In the review of Ross and Densen 3 I I of 77 (I4 %) patients with Ca-deficiency had severe pneumococcal infections, six of them repeatedly. Whether these infections were due to the Ca-deficiency itself or to a related deficient antibody response was not tested. It has been shown by others, however, that an intact classical pathway of complement activation is required for an optimal antibody response to antigen stimulationfl v-29 In guinea-pigs with genetic deficiency of C2 or C4, the antibody response to a T cell-dependent antigen was found to be impaired. 3° In addition, Pryjma and H u m p h r e y 2s showed in decomplemented mice that the response to a T-cellindependent antigen (pneumococcal polysaccharide) is also depressed. In a study of a C4-deficient patient, an impaired humoral immune response due to immaturity of B-cells was suggestedfl I We therefore studied functional antibodies and total concentrations of IgG subclasses in this Ca-deficient patient. Figure I shows that complement was not required for opsonisation of pneumococci in vitro. It was therefore concluded that the complement defect in our patient was not directly responsible for the apparently decreased opsonic capacity for serotype a3-F pneumococci in vivo. In a recent epidemiological study, Bird and Lachmann 32 reported a low concentration of serum IgG4 secondary to inherited deficient complement C3 activation by the classical pathway. Low IgG2 and or IgG4 concentrations have been shown to predispose to recurrent pneumococcal infection. 33' 34 T h e patient's serum had

67

Recurrent f u l m i n a n t pneumococcaemia

indeed concentrations of IgG2 in the lower normal range and decreased concentrations of IgG4 (I6 % of the mean of healthy donors). His sister with the homozygous C2-deficiency, but not his heterozygous mother, also had low concentrations of IgG4. T h e patient's low concentration of IgG4 was not due to an excessive consumption of IgG4 since its decay after infusion of intravenous I g G paralleled decay of concentrations of the other subclasses. Siber et al., 35 observed a correlation between serum IgG2 concentrations and the antibody response to bacterial polysaccharide antigens. These authors showed that even only moderately low IgG2 concentrations are correlated with an impaired response to pneumococcal vaccine. Six m o n t h s after immunisation, 7 o % of the patients with IgG2 < I. 3 g/1 had < 2o0 ng pneumococcal a n t i b o d y - N / m l , whereas only 8 % of the patients with IgG2 > I'3 g/1 had antibodies below this limit. It is therefore conceivable that the moderately low IgG2 concentration of our patient was a further underlying cause for the missing increase of serotype 23-F opsonin after vaccination and natural infection. Since our patient had no underlying malignant disease, the lack of specific antibody response was more likely to be a consequence of his defective humoral i m m u n i t y than of his asplenia per se. 22' 28.25,82 Patients lacking the G 2 M (23) allotype have been shown to have lower IgG2 subclass responses to polysaccharide vaccines. 86'87 T h e combination of C2deficiency, low IgG2 concentrations and the lack of the G2M(23) allotype marker has been previously described. 88 T h e s e combined defects alone, however, do not necessarily result in repetitive infections since our patient's sister with the same inherited defects had an uneventful history. T a k e n together, the recurrent pneumococcaemias were likely to be the result of combined defects since any of t h e m (asplenia, C2-deficiency, IgG2 at a threshold level, and low IgG4 level) predisposes to recurrent pneumococcal infections. Even so, it remains unclear why the patient with congenital defects had the first episode of pneumococcaemia I I years after splenectomy and why the life-threatening infections clustered afterwards. It is conceivable that the first pneumococcal infection paralysed the specific antibody response as shown in animal models. 89' 40 Patients with an impaired specific antibody response to encapsulated bacteria are extremely endangered by splenectomy since the liver is unable to eliminate unopsonised pneumococci. T h u s , after splenectomy, penicillin prophylaxis should be considered for patients with deficiencies of the classical complement pathway. (W. Z. is the recipient of a Career Development Award of the Swiss National Science Foundation (No. 3.8II-o86). Special thanks are addressed to P. J. Lachmann, Cambridge, U.K., and A. G. Sj6holm, Lund, Sweden, for encouraging and very helpful discussions. We thank the staffs of our various institutions for excellent technical help. The results of IgG subclass measurement were kindly confirmed by P. Bird, University of Newcastle-upon-Tyne, U.K. We gratefully acknowledge the secretarial help of Ms Zuzana Kante and Ms Grete V6geli.) Addendum

Since submission of this paper the patient was again admitted to the hospital. A few months after interrupting penicillin prophylaxis against advice, he got 3-2

68

W. Z I M M E R L I ET AL.

abruptly ill with headache, fever and chills. Immediately after the first symptoms the patient took 750 mg amoxycillin. Blood cultures drawn at admission remained sterile, but pneumococcal antigen could be detected in serum on the day of admission and 2 days later. These samples of serum reacted against antibodies from a pool with the antigens 9N, 9V, IoF, I IB, I7F, 23F. With large doses of penicillin he rapidly recovered. Thereafter he started prophylaxis again. References I. Wara DW. Host defense against Streptococcus pneumoniae. The role of the spleen. Rev Infect Dis I98I ; 3: 299-309. 2. Bohnsack JF, Brown EJ. The role of the spleen in resistance to infection. Ann Rev Med I986; 37: 49-59. 3. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency. Medicine I984; 63: 243-273. 4. Schaffner A, Davis CE, Schaffner T, Markert M, Douglas H, Braude AI. In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism. J Clin Invest I986; 78: 5I 1-524. 5. Zimmerli W, Waldvogel FA, Vaudaux P, Nydegger UE. Pathogenesis of a foreign body infection. Description and characteristics of an animal model. J Infect Dis I982; I46: 486-497. 6. Zimmerli W, Seligmann B, Gallin JI. Exudation primes human and guinea pig neutrophils for the subsequent responsiveness to the chemotactic peptide fmet-leu-phe and increases C3bi receptor expression. J Clin Invest I986; 77: 925-933. 7. Joisel F, Leroux-Nicollet I, Lebreton JP, Fontaine M. A hemolytic assay for clinical investigation of human C2. J Immunol Methods I983 ; 59 : 229-235. 8. Harrison RA, Lachmann PJ. Complement technology. In: Weir DM, Ed. Handbook of Experimental Immunology, chapter 39. Oxford: Blackwell Scientific, I986. 9. Gold ER, Fundenberg HM. Chronic chloride. A coupling reagent for passive hemagglutination reactions. J Immunol I967; 99: 859-866. IO. Spaeth PJ, Corvetta A, Nydegger UE, Montront M, Bfitler R. An extended CIq-binding assay using lacto-peroxidase- and chloramine-T-iodinated Cxq. Scand J Immunol I983; I8: 319-328. I I. King H, Schumaker HB Jr. Splenic studies. I. Susceptibility to infection after splenectomy performed in infancy. Ann Surg I952; *36: 239-242. I2. O'Neal BJ, McDonald JC. The risk of sepsis in the asplenic adult. Ann Surg I98I ; *94: 775-778. I3. Schwartz PE, Sterioff S, Mucha P, Melton I I I LJ, Offord KP. Postsplenectomy sepsis and mortality in adults. J A M A i982; 248: 2279-2283. I4. Ludmerer KM, Kissane JM. Septic shock in a young splenectomized man. Am J Med I983; 74: I29-I43. I5. ChaikofEL, McCabe CJ. Fatal overwhelming postsplenectomyinfection. A m J S u r g I985; I49 : 534-539. I6. Horan M, Colebatch FH. Relation between splenectomy and subsequent infection. A clinical study. Arch Dis Child I962; 37: 398-414. 17. Donaldson S S, Moore MR, Rosenberg SA, Vosti KL. Characterization of postsplenectomy bacteremia among patients with and without Iymphoma. N Engl J Med I972; 287:69-7 I. I8. Singer DB. Postsplenectomy sepsis. Perspect Pediatr Pathol I973; *: 285-3II. I9. Hosea SW, Butch CG, Brown EJ, Berg RA, Frank MM. Impaired immune response of splenectomized patients to polyvalent pneumococcal vaccine. Lancet I98I ; i: 804-8o7. 2o. Winkelstein JA, Lambert GH. Pneumococcal serum opsonizing activity in splenectomized children. J Pediatr I975 ; 87 : 430-433 • 2I. Nielsen JC, Buskjaer L, Lamm LU, Soiling J, Ellegaard J. Complement studies in splenectomized patients. Scand J Haematol I983 ; 3o : I94-2oo.

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