Data collected through a proforma (Appendix IV) were entered into SPSS version 16. Percentages were used to describe frequency analysis of categorical variables. Numerical variables summarized as means and standard deviations where appropriate. Pearson’s chi-square was used to determine associations between categorical variables. Means of numerical variables were compared within categorical variables using 2-sample independent t-test. A p-value of <0.05 defined the level of statistical significance.
CHAPTER FIVE RESULTS DEMOGRAPHICS
One hundred and sixteen HIV-Infected children and sixty one (61) age and sex-matched HIV-negative controls whose parents and or care-givers consented were recruited for this study. Both subjects and controls ages ranged from 18 months to 14 years with mean age being 7.2 ± 3.3 years and 8.7± 3.1 years, respectively.
Of the 116 subjects, 63 (54.3%) were males and 53 (45.7%) were females and the controls consisted of 26 (42.6%) males and 35 (57.4%) females. A hundred and four of the subjects (89.7%) were on highly active antiretroviral therapy (HAART) of varying regimens. Ninety seven of the 104 were on Zidovudine-based fixed drug combination (FDC), 4 were on Abacavir, Lamivudine and Zidovudine, 3 were on Lamivudine, Zidovudine and Efavirenz and 12 (10.3%) were HAART-nạve.
Eleven (84.6%) of the subjects who were found to be positive for antiphospholipid antibodies had been on HAART for more than 6 months.
Nine were in World Health Organization (WHO) clinical stage 1 and two were in stages 2 and 3. None of the subjects had any symptom or history suggestive of autoimmunity in the past or at recruitment.
LABORATORY PARAMETERS CD4+ LYMPHOCYTE COUNT
The CD4+ lymphocyte count of the subjects and controls ranged from 5 cells/µl to 2,169 cells/µl and 562 cells/µl to 2,192 cells/µl respectively. The mean (±SD) CD4+ lymphocyte count for subjects and controls were 878 ± 541
cells/µl and 1,178 ± 327 cells/µl respectively and the difference was statistically significant (p value = 0.0002) as shown in Table 5.1. However, two of the 116 subjects had counts less than 350 cells/µl and none of the controls had CD4+ cell count less than 350 cells/µl.
FULL BLOOD COUNT
Haematocrit: values amongst the subjects and controls ranged from 16% to 40.4% and 30.2% to 45.7% respectively. The mean values being 33.6 ± 3.7%
and 37 ± 3.5% for subjects and controls respectively and the difference was significant (p < 0.0001) as shown in Table 5.1.
White Blood Cell Count: The values obtained amongst subjects ranged from 1.3 to 16.5 x 109/L and mean ± SD was 5.6 ± 2.9 x 109/L. The results among the 61 controls had values ranging from 2.1 to 21.9 x 109/L (mean: 4.9 ± 3.2 x 109/L). The difference was not statistically significant (p= 0.2). Leucopenia was not observed in either the subjects or the controls that tested positive for antiphospholipid antibodies.
Platelet count: The mean (± SD) platelet count value for all subjects was 268
± 104 x 109/L while for the 61 controls the mean (± SD) was 219 ± 79 x 109/L.
The difference was statistically significant with p value being 0.002.
Thrombocytopaenia was not observed in subjects and controls who were positive for antiphospholipid antibodies (aPLs) as their platelet count values ranged from 146 to 407 x 109/L and 100 to 415 x 109/L respectively.
Two -sample independent t-test was done to determine the effect of antiphospholipid antibody positivity or negativity within the subject group on
the PCV, Platelet and White cell counts respectively and was found not be significant (p-values being 0.41, 0.82 and 0.66 respectively). Table 5.3
DIRECT COOMBS’ TEST: This was negative in all subjects whether antiphospholipid antibodies positive or not. Controls also showed negative results.
ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)
Nineteen (19) childrens’ citrated plasma samples were pulled and used as control. This pulled control plasma had a mean aPTT value of 37.3 secs.
Subject specimen with aPTT value 7 seconds more than 37.3 secs i.e 44.3 secs was taken as prolonged results.
Amongst the 116 subjects, the mean (±SD) value for aPTT was 39.4 ± 4.2 secs with a range between 28.8 seconds and 54.4 sec. Thirteen of the subjects had prolonged values ( > 44.3 secs) ranging between 44.3 and 54.0 secs. Mixing test was done on these 13 samples and did not correct the prolonged values (> 44.3 secs). These 13 samples were further screened for Lupus anticoagulant.
The aPTT values of the control patients ranged from 34.5 sec to 57.0 sec with a mean (± SD) of 40.1 ± 4.0. Similarly, nine of the controls had prolonged aPTT values (>44.3secs) and when the mixing test was carried out, eight samples still had prolonged values. These eight samples were screened further for LA. There was no statistically significant difference between the mean aPTT values of both subjects and controls (p = 0.3) Table 5.1.
However, there was significance when mean aPTT values were compared
between subjects and controls positive for antiphospholipid antibodies (aPLs) and those negative with p value of 0.0000. Table 5.3
ANTIPHOSPHOLIPID ANTIBODY SCREENING
Two antiphospholipid antibodies (aPLs) were screened for; (1) Lupus anticoagulant (LA) as determined by kaolin clotting time (KCT) test and (2) Anticardiolipin antibody (aCL) determined by serological test.
KAOLIN CLOTTING TIME (KCT)
Kaolin clotting time and anticardiolipin assay were carried out on 13 subjects and 8 control samples.
Seven (6.03%) of 116 subjects and 4 (6.56%) of the 61 control group were positive for lupus anticoagulant (i.e KCT-R ≥ 1.2). LA was not more prevalent among subjects compared with the controls (p =0.89). Of the 7 subjects that were LA positive, four were (i.e 4 of 63 males) and 3 were females (i.e 3 of 53 females) were positive for Lupus anticoagulant. Sex within the subject group had no association with development of LA (p =0.87).
Similarly, within the control group, two males were LA positive (i.e 2 out of 26) and two females (i.e 2 out of 35) showed positivity for lupus anticoagulant.
Furthermore, sex was found not to be associated with LA development (p = 0.75). The mean ages for LA positivity in subjects and controls were 5.9 ± 1.9 years and 9.75 ± 2.7 years respectively and the difference was not significant (p > 0.05). Six of the 104 subjects on HAART were positive for LA while one of 12 HAART-nạve subjects was positive.
ANTICARDIOLIPIN ANTIBODY TEST (IgG/IgM)
Eleven (9.48%) of the 116 subjects were positive for anticardiolipin antibodies and 6 (9.84%) of the 61 control group were also positive for anticardiolipin antibodies. Anticardiolipin antibody was found not to be more prevalent among the subjects compared with the controls (p = 0.93). Of the 11 subjects who were positive for aCL, 6 were males (i.e 6 of 63 males) 5 were females (i.e 5 of 53 females). Sex did not impact on the development of anticardiolipin antibodies (p = 0.98).
Within the control group, 3 males were for aCL (i.e 3 of 26 males) and 3 females were also positive (i.e 3 of 35 females). Similarly, sex was not associated with the development of anticardiolipin antibodies among the controls (p = 0.70).
Of the 11 subjects that were positive for aCL, 8 were positive for both Ig G and Ig M. Two were positive for Ig G only and one was positive for Ig M only.
Of the six people that were positive in the control group, 3 were positive for both Ig G and Ig M. The rest were positive for Ig M only.
Low positive titres were observed among 8 of the subjects and 4 of the controls. Mean age of subjects positive for aCL was 8.0 ± 1.8 years while the mean age of controls positive for aCL was 9.1 ± 2.5 years and this difference was not statistically significant (p > 0.05).
Overall in the subject group, there were 5 patients that were positive for both aCL and LA, there were 6 patients that were positive for aCL only and there were 2 patients that were positive for LA only. Thus, the total number of antiphospholipid antibody detected in the subject group is thirteen. Therefore, prevalence of aPL in the subject group is 11.2% (13 of 116). Of these 13
positives, 11 occurred in the HAART-experienced HIV-infected subjects (104) and 2 occurred among the 12 HAART-nạve subjects.
The prevalence of antiphospholipid antibodies (aPLs) was compared between patients on HAART (11 of 104) and HAART-nạve patients (2 of 12).
No association was observed between those on HAART and HAART-nạve for the development of aPLs ( =0.4, p =0.52). Additional finding was that HIV infection did not predispose to having antiphospholipid antibodies (aPLS) as 13 of 116 HIV-infected patients had aPLs and 6 of 61 HIV negative patients had aPLs ( =0.49, p =0.78).
Table 5.1: Demographic characteristics and laboratory parameters (mean ±SD) of subjects and controls
Parameters Subjects(n= 116 ) Controls (n =61) p value Gender Male = 63 (54.3%) Male = 26 (42.6%) 0.0001 Female = 53 (45.7%) Female = 35 (57.4%)
Age (years) 7.2 ± 3.3 (median = 7) 8.8 ± 3.1 (median = 9) CD4+ (cells/µl) 878 ± 541 1,178 ± 327 0.0002 PCV (%) 33.6 ± 3.7 37.5 ± 3.5 < 0.0001 WBC (x 109/L) 5.6 ± 2.9 4.9 ± 3.2 0.2 Platelet Count (x 109/L) 268 ± 104 219 ± 79 0.002 APTT (sec) 39.4 ± 4.2 40.1 ± 4.0 0.3 Legends: CD4+: CD4 + lymphocyte count; PCV: packed cell volume; WBC:
white blood cell count; PLT: platelet count; APTT: activated partial thromboplastin time
Table 5.2: Haematological profile of subjects and controls positive for antiphospholipid antibodies
Parameters Subjects (n = 13) Controls (n = 6) t value p value PCV (%) 32.9 ± 3.2 37.3 ± 3.3 3.3 0.004
WBC (109/L) 5.7 ± 2.5 4.1 ± 713 -2.1 0.05 PLT (109/L) 302 ±109 235 ± 119 -1.3 0.1 CD4+ (cells/µl) 964 ± 817 1,200 ± 284 1.2 0.2 APTT (sec) 43.4 ± 5.7 45.6 ± 5.3 0.86 0.39
Legends: PCV: packed cell volume; PLT: platelet count; CD4+: CD4 +
lymphocyte count; APTT: activated partial thromboplastin time
Table 5.3: Comparison of the effect of antiphospholipid antibody positivity and negativity on haematological variables.
aPL: antiphospholipid antibodies, POS: positive, NEG: negative, PCV: packed cell volume, WBC: white cell count, PLT: platelet, CD4: cluster of differentiation 4, APTT: activated partial thromboplastin time
Subjects Controls
PCV(%) aPL POS aPL NEG t-test p-value Mean ±SD 32.7 ± 4.1 33.7 ± 3.6 0.84 0.41
WBC (x 109/L) 5,845 ± 2,541 5,635 ± 3,026 -0.23 0.82
PLT (x 109/L) 280,333 ± 85,970 265,913 ± 107423 -0.44 0.66
CD4 (/µl) 812 ± 557 885 ± 542 0.43 0.67
APTT (secs) 46.6 ± 3.9 38.4 ± 3.2 -8.45 0.0000
aPL POS aPL NEG t-test p-value Mean ±SD 37.4 ± 3.3 37.5 ± 3.5 0.04 0.97
4,171 ± 713 5,099 ± 3,475 0.69 0.49
235,000 ± 119,983 216,880 ± 73,442 -0.56 0.58
1,248 ± 284 1,168 ± 333 -0.61 0.55
45.6 ± 5.3 39.3 ± 3.2 -4.48 0.0000
Table 5.4: Subjects and controls with positivity for aCL and titres of Ig G and Ig M
Subjects Ig G Ig M 6 High Positive High Positive 7 Low Positive Low Positive 13
17 18 19 27 80 104 113 117 Controls
C6 C15 C35 C43 C53 C58
Low Positive Low Positive Low Positive Low Positive Moderate Positive Low Positive Low Positive Negative Low Positive
Ig G
Low Positive Low Positive Negative Negative Negative Low Positive
Low Positive Moderate positive Low Positive Negative Low Positive Low Positive Negative Low Positive Low Positive
Ig M
Low Positive Low Positive Low Positive Low Positive Moderate Positive Moderate positive
Legends: Ig – Immunoglobulin; aCL anticardiolipin antibody
Figure 5.1: Number of male and female subjects and controls positive for Lupus anticoagulant.
0 1 2 3 4 5 6 7 8
Positivity Male Female
Lupus Anticoagulant
Subjects Control
Figure 5.2: Subjects positive for anticardiolipin antibodies Ig G and M.
0 2 4 6 8 10 12
IgG IgM Both
Anticardiolipin Antibodies (Subjects)
Positivity Male Female
Figure 5.3: Controls positive for anticardiolipin antibodies Ig G and M.
0 1 2 3 4 5 6 7
IgG IgM Both
Anticardiolipin Antibodies (Control)
Positivity Male Female
CHAPTER SIX DISCUSSION
Infection and autoimmune disorders have been implicated in the development of antiphospholipid antibodies in children. Some of the common infectious triggers include viral, bacterial, parasitic and spirochaetal, however, viral infections are the commonest. 58,70,71 Antiphospholipid antibodies triggered by infections have been described as being transient and non pathogenic.71 The antiphospholipid antibodies most frequently found are the lupus anticoagulant (LA) and anticardiolipin antibodies (aCL).72
In this study, 11.2% (13 out of 116) of HIV-infected children were positive for antiphospholipid antibodies compared to 9.8% (6 out of 61) HIV negative controls and this is consistent with findings by Shah et al.12,85
The only study done among children in Nigeria was by Olayemi et al in sickle cell disease patients who found a prevalence of 1.8% for LA.21 Although sickle cell disease is also considered an immunosuppressive disorder, the degree of immunosuppression is not comparable to that obtained in HIV infection; in which patients are susceptible to bacterial, viral, fungal and parasitic infections that may trigger the formation of antiphospholipid antibodies.
It is not surprising that 9.8% of the HIV-negative controls were also positive for aPLs since it is not only HIV infection that can trigger the formation of these antibodies.35,36
Moreover, the prevalence of anticardiolipin antibodies among healthy children have been reported to be between 2% and 82% in some studies.73,74,75 With regards to the prevalence of aPLs in HIV- infected
children, the literature is sparse with most reports being incidental findings and case reports.12,85
A higher prevalence rate of lupus anticoagulant was observed in this study compared to similar studies amongst adult HIV-infected patients.19,23 This could be due to the methods used in this study. The use of two various methods increases the sensitivity and specificity of results.
Of the two subtypes of antiphospholipid antibodies (LA and aCL) tested for in this study, anticardiolipin antibodies (aCL) were found to be higher than lupus anticoagulant. The association of aCL with HIV infection was first reported about 23 years ago among male homosexuals.62 A year later Coll et al77 alsoreported a prevalence of 59.5% aCL IgG among 84 HIV patients.
The IgG isotype was higher in the subjects in this study and similar finding was reported by Stimmler et al78 in 26 adult HIV-infected patients in whom a higher mean of IgG aCL was observed. However, these had AIDS and opportunistic infections (OIs) but interestingly, none of the subjects positive for anticardiolipin antibodies in this study had AIDS or OIs.
In HIV infection, both types of aCL (the non-pathogenic or β2 -GP1-independent) and the pathogenic (β2-GP1-dependent) may be detected and this may influence the clinical presentations that can be seen.
Anticardiolipin antibodies were similarly found at a higher frequency among controls in this study with aCL IgM being significantly higher than IgG isotype (100% vs 42.9%). The finding of a higher aCL positivity among this healthy controls in this study was similar to findings in other studies.73,74,75However, works by Rapizzi et al74 (26% vs 1%) and Galrao et al79 (16.7% vs 3.3%) revealed a higher IgG isotype and this may be
considered as the epiphenomenon of vaccinations, nutrition and infections in the paediatric population.75,80
Clinically all subjects were asymptomatic and belonged to either WHO Clinical stage 1 (100), 2 (10) and 3 (6). Mean CD4+ lymphocyte count among the subjects was 971cells/µl and eleven of the subjects who showed positivity for aPLs had CD4 + counts above 350cells/µl while 2 had counts less than 350cells/µl (24cells/µl and 161cells/µl but they had no symptoms of AIDS.
Likewise, 11 were also on Highly active antiretroviral therapy (HAART) and two were HAART-nạve and their naivity was because they had not completed the necessary pretherapy investigations.
A study by Ankri et al81 among 90 HIV-infected patients found a lower CD4+ lymphocyte count in patients with antiphospholipid syndrome (APS) compared to those without APS and that gender, risk factors and stage of disease between patients with HIV and IgG aCL and HIV-infected patients without antibody was not significant.
No symptom suggestive of autoimmunity or peculiar to the APS was found in the subjects. Generally, it has been said that antibodies induced by infections are transient, of low titre, non-pathogenic and most often of the IgM subtype however, several reports have shown otherwise.35,38,71,83
Low-positive titres of aPL found in this study agrees witn the description of aPL induced by infective organisms and this explains the absence of any clinical feature related to the presence of aPLs.
Clinical manifestations due to the presence of these antibodies vary among studies. It has been said that because of the relatively low prevalence of APS and its heterogeneity among children, systematic studies are sparse.82
A “two-hit” hypothesis has been proposed to explain the presence of high aPL titres and the absence of symptoms. This “two-hit” hypothesis states that the presence of aPLs induces endothelial dysfunction(“first-hit”) and then another condition which may be pregnancy, infection, smoking, hypertension, atherosclerosis, obesity or vascular injury (second-hit) triggers thrombosis.2,84 However, most of the “second-hits” are not known risk factors in children. The concept of developmental haemostasis which states that there are age-dependent differences in the coagulation and fibrinolytic system can explain the absence of these features. In addition to this the relatively healthy state of the vascular endothelium can play a role.85
Moreover, Hunt et al37 observed that the incidence of thrombosis is lower in children than in the adults because of increased levels of physiologic anticoagulants.
The occurrence of thrombosis either arterial and or venous has been reported with antiphospholipid antibodies, though of low incidence in children.12,37,58
Shah et al12,86 have reported a case of thrombosis and recurrent thrombosis in children. Reduced CD4+ lymphocyte counts (<200cells/µl), presence of opportunistic infections, neoplasms, or autoimmune disorder such as autoimmune haemolytic anaemia may predispose to thrombosis.
Seaman et al87 found a significant association of aCL with thrombotic events. Also, a strong relationship between the presence of LA and thrombotic events was found by Berube et al88 who observed that having both LA and aCL did not strengthen the relationship between patients with thrombotic complications and aPLs.90 This was also observed in this study as
none of those who were positive for both LA and aCL presented with or gave history suggestive of thrombosis. Ravelli et al89 found aCL to have a low predictive value for the development of thrombosis
Anaemia was the only observed cytopaenia in the subjects who were positive for antiphospholipid antibodies (aPLs) and it was found not to be due to the presence of these antibodies but due to the HIV infection. Also, the anaemia seen was not haemolytic even though this is more commonly seen in patients. Thrombocytopenia has been reported to occur in 20 – 40% of patients with APS was not documented in this study.10
Direct Coombs’ test was negative in all subjects. This is similar to what Ndakotsu et al23 found in their study among adults patients. This may be due to the dysfunctional state of the immune system in HIV infection and/ or immature nature of the immune system in children.
CHAPTER SEVEN CONCLUSION
This study showed that antiphospholipid antibodies (LA and aCL) can be found in HIV-infected or negative children. Anticardiolipin antibody was found to be more prevalent (11 of 116) as compared with LA (6 of 61) although this is not of statistical significance.
The study also showed that the features associated with the presence of these antibodies or antiphospholipid syndrome were not observed and that cytopaenia observed were mild and not due to the presence of these antibodies.
HIV infection did not determine the development of antiphospholipid antibodies. Similarly, antiphospholipid antibody positivity was not influenced by being on HAART or being HAART-nạve.
LIMITATIONS
The high cost of reagents made it difficult to do the assay longitudinally. The tests carried out to detect these antibodies were done once as such this made it impossible to truly determine if they were transient or not because a positive finding after 12 weeks may suggest a tendency to having the antiphospholipid syndrome.
RECOMMENDATIONS
Routine tests for the detection of antiphospholipid antibodies may not be needed in HIV-infected and apparently healthy children but should be
considered in children with high activated partial thromboplastin time (aPTT) value.
REFERENCES
1. Zandman-Goddard G, Shoenfeld Y. HIV and autoimmunity.
Autoimmunity Reviews 2002; 1: 329-337.
2. Levine JS, Branch DW, Rauch J. The Antiphospholipid Syndrome. N Engl J Med: 2002; 346:752-763.
3. Hanly JG. Antiphospholipid Syndrome: an overview. CMAJ 2003; 168 (13): 1675-1682.
4. Olayemi E, Halim NKD. Antiphospholipid Antibodies in Medical Practice:
A review. Nig J Medicine 2006; 15(1): 7-15.
5. Galli M, Comfurius P, Barbui T, Zwaal RFA, Bevers EM. Anticoagulant activity of β2- glycoprotein 1 is potentiated by a distinct subgroup of anticardiolipin antibodies. Thromb Haemost 1992; 68: 297-300.
6. Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome; a systematic review of the literature. Blood 2003; 101(5): 1827-1832.
7. Tektonidou M. Antiphospholipid Syndrome. Orphanet encyclopedia 2004: 1-6.
8. Brew BJ, Miller J. Human Immunodeficiency virus type1-related transient neurological deficits. Am J Med 1996; 18: 380-391.
9. Avcin T. Antiphospholipid syndrome in children. Curr Opin Rheumatol 2008; 20: 595-600.
10. Saigal R, Kansal A, Miltal M, Singh Y, Ram H. Antiphospholipid Antibody syndrome. J Assoc Physicians India. 2010: 58:176-184.
11. Roddy SM, Giang DW. Antiphopholipid antibodies and stroke in an infant. Pediatrics 1991; 87: 933-935.
12. Shah I, Cudgel P. Antiphospholipid syndrome in a Human Immunodeficiency virus 1-infected child. Pediatr Infect Dis J, 2006; 25(2):
185-186.
13. Kamat AV, D’Cruz DP, Hunt BJ. Managing antiphospholipid antibodies and antiphospholipid syndrome in children. Haematologica 2006; 91:
1674-1680.
14. Blank M, Krause J, Fridkin M, Keller N, Kopolovic J, Goldberg I, et al.
Bacterial induction of autoantibodies to beta 2-glycoprotein-1 accounting for infectious etiology of antiphospholipid syndrome. J Clin Invest 2002;
109: 797-804.
15. De Larranaga GF, Forastiero RR, Carreras LO, Alonso BS. Different types of Antiphospholipid Antibodies in AIDS: A comparison with syphilis and the Antiphospholipid syndrome. Thrombo Res 1999; 96: 19-25.
16. Kopelman RG, Zolla-Pazner S. Association of human immunodeficiency virus infection and autoimmune phenomena. Am J. Med 1988; 84: 82-88.
17. Awodu OA, Shokunbi WA, Ejele OA. Lupus anticoagulant in Nigerian women with preeclampsia. West Afr J. Med 2003; 22: 240-242.
18. Awodu OA, Ejele OA, Shokunbi WA, Enosolease MA. Prevalence of Lupus Anticoagulant in multiparous women in Benin City, Nigeria. Niger Postgrad Med. J. 2003; 10:19-22.
19. Awodu OA., Olayemi EE, Bazuaye GN, Onunu AN. Lupus anticoagulant, in human immunodeficiency virus-infected patients on
highly active antiretroviral therapy. Indian J of Pathol Microbiol 2010; 53 (1): 47-49.
20. Olayemi E. Prevalence of Lupus Anticoagulant in Sickle cell Disease Patients in Benin City. WACP dissertation. West African College of Physicians. April 2004.
21. Olayemi E, Halim NKD, Durosinmi MA, Awodu OA. Lupus Anticoagulant in Nigerian Children with Homozygous sickle cell disease. Annals of African Medicine. 2005; 4(3): 122-126.
22. Akinbami AA, Akanmu AS. Afolabi BB, Wright KO, Dada MO., Dosunmu AO. Prevalence of Antiphospholipid Antibodies in Pregnant Women at a Secondary health Care Institution in Lagos. The Internet Journal of Hematology; 2009: 6(1). www.ispub.com. Date Accessed 5/3/2011
23. Ndakotsu MA, Salawu L, Durosinmi MA. Lupus anticoagulant in Nigerian patients living with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome. J Microbiol Immunol Infect. 2009; 42: 69-73.
24. Kotila TR, Fasola FA. The Lupus Anticoagulant in a population of Healthy Nigerian Adults. Annals of Ibadan Postgraduate Medicine. 2005;
3 (1); 45-48.
25. Wassermann A, Neisser A, Bruck C. Eine Serodiagnostiche Reaktion bei Syphilis. Deutsche Med Wochenschr 1906; 32: 745-746.
26. Pangborn MC. A new serologically active phospholipid from beef heart.
Proc. Soc. Exp. Biol Med 1941; 48: 484-486.
27. Viard JP, Amoura Z, Bach JF. Association of anti-2–glycoprotein-1 antibodies with lupus-type circulating anticoagulant and thrombosis in systemic lupus erythematosus. Am J Med 1992; 93:181-186.
28. Conley CL, Hartman RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest, 1952; 31:621.
29. Laurell A, Nilsson I. Hypergammaglobulinemia and biological false positive Wassermann reaction: a study of two cases. J Lab Clin Med, 1957;49: 694.
30. Lee SL, Saunders MA. A disorder of blood coagulation in SLE. J Clin Invest. 1995; 34; 181.
31. Harris EN, Gharavi AE, Boey ML, Patel BM, Mackworth-Young CG, Loizou S et al. Anticardiolipin antibodies: by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983; 2: 1211-1214.
32. Mc Neil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid- binding inhibitor of coagulation. Beta 2 - glycoprotein 1(apolipoprotein H).
Pro Natl Acad USA 1990; 87: 4120-4124.
33. Hunt JE, Mc Neil HP, Morgan GJ, Crameri RM, Krilis S. A phospholipid beta-2-glycoprotein 1 complex is an antigen for anticardiolipin antibodies occurring in autoimmune disease but not with infection. Lupus 1992; 1:
75-81.
34. Petri M. Epidemiology of the antiphospholipid antibody syndrome. J Autoimmun 2000; 15:145-51.
35. Kurugol Z, Vardar F, Ozkinay F, Kavakli K, Ozkinay C. Lupus anticoagulant and protein S deficiency in otherwise healthy children with acute varicella infection. Acta Paediatr 2000; 89: 1186-1189.
36. Briones M, Abshire T. Lupus anticoagulants in children. Curr Opin Hem 2003; 10: 375-379.
37. Hunt BJ. Pediatric Antiphospholipid antibodies and Antiphospholipid syndrome. Semin Thromb Hemost 2008; 34: 274-281.
38. Male C, Lechner K, Eichinger S, Kyrle PA, Kapiotis S, Wank H et al.
Clinical significance of lupus anticoagulants in children. J Pediatr 1999;
134: 199-205.
39. Gallistl S, Munfean W, Leschnik B, Meyers W. Longer aPPT values in healthy children than in adults; no single cause. Thromb Res 1997; 88:
355-359
40. Von Landerberg P, Lehman HW, Knoll A, Dorsch S, Modrow S.
Antiphospholipid antibodies in pediatic and adult patients with rheumatic disease are associated with parvovirus B19 infection. Arthritis Rheum 2003; 48: 1939-1947.
41. Cervera R, Khamashta MA, Font J. Systemic lupus erythematosus:
clinical and immunologic patterns of disease expression in a cohort of 1,000 patients. Medicine (Baltimore) 1993: 72:113-124.
42. Love PE, Santoro SA. Antiphopholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorder; Prevalence and clinical significance. Ann Intern Med 1990;
112:682-698.
43. Khamashta MA, Cuadrado MJ, Mujic F, Taub NA, Hunt BJ, Hughes GR.
The management of thrombosis in the antiphospholipid-antibody syndrome. N Eng J Med 1995; 332: 993-997.