Algorithm of differential diagnosis of anemia involving laboratory medicine specialists to advance diagnostic excellence.

OBJECTIVES: Anemia is a severe global public health issue. Testing practices for anemia suggest overuse of screening laboratory tests and misinterpretation of studies even in "easy-to-diagnose" underlying causes, leading to late diagnoses and missed treatment opportunities. We aimed to develop a complete and efficient algorithm for clinical pathologists and laboratory medicine physicians for the differential diagnosis of anemia. METHODS: Comprehensive literature search encompassing original articles, studies, reviews, gold standard books, and other evidence. RESULTS: We created a complex algorithm, primarily for clinical pathology/laboratory use, that explores all major and several rare causes of anemia in an efficient and evidence-based manner. The algorithm includes gold-standard diagnostic laboratory tests available in most clinical laboratories and indices that can be easily calculated to provide an evidence-based differential diagnosis of anemia. CONCLUSIONS: The diagnostic strategy combines previously available diagnostic tests and protocols in an efficient order. Clinical pathologists following the algorithm can independently provide valuable diagnostic support for healthcare providers. Clinical pathologists providing complete differential diagnostic services with the proposed algorithm may create an opportunity for an advanced diagnostic service that supports diagnostic excellence and helps patients receive a timely diagnosis and early treatment opportunities.

An international study on activated partial thromboplastin time prolongation. Part 2: Interpretative commenting.

BACKGROUND: Providing evidence-based interpretative comments (IC) is an integral task of clinical laboratory professionals. It may be of special relevance for coagulation testing, where pathological first-line tests could trigger more specialized tests. Our aim was to evaluate the quality of ICs provided to the physician in two samples with activated partial thromboplastin time (APTT) prolongation. MATERIAL AND METHODS: Two lyophilized plasma samples and their respective fictional clinical cases (case 1: heparin contamination and case 2: factor VIII deficiency) were sent to European laboratories for APTT and APTT mixing test measurement, and elaboration of ICs based on their results. The quality of ICs was evaluated in terms of analytical classification, laboratory interpretation, advice to physician, clarity, length and whether the clinical question was answered. RESULTS: A total of 214 laboratories were included. Classification of the analytical result was stated in 57 % of comments. Laboratory interpretation was found in 91 % of comments for case 1 and 83.3 % for case 2, among which 9.3 % and 6.5 % were considered wrong, respectively. Advice for the requesting physician was provided in 65.8 % of comments for case 1 and 61.2 % for case 2, among which 36 % and 4.7 % were considered wrong, respectively. More than 70 % of comments for both cases were evaluated as clear and of an adequate length. CONCLUSION: A significant number of laboratories provide clear interpretations and helpful advice for the management of altered coagulation results. Nevertheless, the finding of several confusing and misleading comments highlights the need for recommendations on elaboration of interpretative comments.

An international study on activated partial thromboplastin time prolongation. Part 1: Analytical results.

BACKGROUND: Unexpected prolongation of first-line coagulation tests, including activated partial thromboplastin time (APTT), should trigger further work-up by performing mixing tests to elucidate the underlying cause, direct further specific testing and clarify their possible clinical impact. The aim of our study was to assess whether methodological diversity has any impact on the APTT mixing test results and their interpretation. MATERIAL AND METHODS: Two lyophilized plasma samples (case 1: heparin contamination [0.5 IU/mL]; case 2: factor VIII deficiency [0.13 IU/mL]) and their respective fictional clinical cases were sent to European laboratories for APTT measurement and performance of mixing tests. Participants were surveyed about the methodology (reagents, analytical platform, reference ranges), APTT results, mixing test conditions, their classification (normal, equivocal, prolonged) and categorization of the sample (factor deficiency, presence of inhibitor, anticoagulant, unknown). RESULTS: A total of 269 responses were included. For case 1, all participants reported a prolonged APTT, and 91% obtained no correction in the mixing test, without differences among reagents or analytical platforms. Only 15% of them selected the presence of an anticoagulant as the single cause for the prolongation. For case 2, 99% of participants reported a prolonged APTT, while some heterogeneity in the mixing test results was found. Eighty-six percent of participants selected factor deficiency as the cause for APTT prolongation. CONCLUSIONS: Most European laboratories obtained valid results for APTT and the subsequent mixing tests, despite using different methodologies. However, their classification could be improved. Therefore, more training and periodic evaluations are recommended to harmonize protocols and ensure proper result classification and categorization.

Immune response against the severe acute respiratory syndrome coronavirus 2 / A súlyos akut légzoszervi szindrómát okozó koronavírus-2 elleni immunválasz

Coronavirus disease 2019 (COVID-19) displays tremendous inter-individual variability, ranging from asymptomatic infections to life-threatening illness. Although more studies are needed, a picture has begun to emerge that variability in the immune system components is a main contributor to the heterogeneous disease courses. Here, we provide a concept for the interaction of the innate and adaptive immune systems with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to link the observations that have been made during the first two years of the pandemic. Inborn errors of, and autoantibodies directed against, type I interferons, dysregulated myeloid response, hyperinflammation, lymphopenia, lymphocyte impairment, and heterogeneous adaptive immunity to SARS-CoV-2 are discussed, as well as their impact in the course of COVID-19. In addition, we will also review part of the key findings that have helped define and delineate some of the essential attributes of SARS-CoV-2-specific humoral and cell -mediated immune memory.

Antibody and T Cell Responses against SARS-CoV-2 Elicited by the Third Dose of BBIBP-CorV (Sinopharm) and BNT162b2 (Pfizer-BioNTech) Vaccines Using a Homologous or Heterologous Booster Vaccination Strategy.

In the present study, antibody and T cell-mediated immune responses elicited by BBIBP-CorV and BNT162b2 vaccines were compared 6 months after the two-dose immunization of healthy individuals. Additionally, antibody and T cell responses after the third dose of BBIBP-CorV or BNT162b2 were compared using a homologous or heterologous vaccination strategy. The third dose was consistently administered 6 months after the second dose. Six months following the two-dose vaccination, the cumulative IFNγ-positive T cell response was almost identical in participants immunized with either two doses of BNT162b2 or BBIBP-CorV vaccines; however, significant differences were revealed regarding humoral immunity: the two-dose BNT162b2 vaccine maintained a significantly higher antireceptor-binding domain (RBD) IgG, anti-spike (S1/S2) IgG, and IgA antibody levels. The BNT162b2 + BNT162b2 + BBIBP-CorV vaccine series elicited significantly lower anti-RBD IgG and anti-S1/S2 IgG levels than three doses of BNT162b2, while the anti-S IgA level was equally negligible in both groups. Importantly, the cumulative IFNγ-positive T cell response was highly similar in both groups. Surprisingly, the BBIBP-CorV + BBIBP-CorV + BNT162b2 vaccination series provided a much higher cumulative IFNγ-positive T cell response than that elicited by three doses of BNT162b2; moreover, the levels of anti-RBD IgG and anti-S IgA were almost identical. Only the mean anti-S1/S2 IgG levels were higher after receiving three mRNA vaccines. Based on these data, we can conclude that administering a third dose of BNT162b2 after two doses of BBIBP-CorV is an effective strategy to significantly enhance both humoral and T cell-mediated immune response, and its effectiveness is comparable to that of three BNT162b2 vaccines.

Comparison of antibody and T cell responses elicited by BBIBP-CorV (Sinopharm) and BNT162b2 (Pfizer-BioNTech) vaccines against SARS-CoV-2 in healthy adult humans.

In the present study, humoral and T cell-mediated immune responses elicited by BBIBP-CorV (inactivated virus) and BNT162b2 (mRNA-based) vaccines against SARS-CoV-2 virus were compared. Convalescent volunteers were also investigated to evaluate adaptive immunity induced by live virus. Although both vaccines induced antibody- and T cell-mediated immune responses, our analysis revealed significant quantitative and qualitative differences between the two types of challenges. The BBIBP-CorV vaccine elicited antireceptor-binding domain (RBD) IgG, as well as anti-spike protein (S) IgG and IgA antibodies in healthy individuals, the levels of which were much lower than after BNT162b2 vaccination but still higher than in the convalescent patients. The cumulative IFNγ-positive T cell response, however, was only twofold higher in participants injected with BNT162b2 compared to those who were primed and boosted with BBIBP-CorV vaccine. Moreover, the inactivated virus vaccine induced T cell response that targets not only the S but also the nucleocapsid (N) and membrane (M) proteins, whereas the mRNA vaccine was able to elicit a much narrower response that targets the S protein epitopes only. Thus, the pattern of BBIBP-CorV-induced T cell response in virus-naive participants was similar to the cell-mediated anti-SARS-CoV-2 response observed in convalescent patients. Based on these data, we can conclude that the BBIBP-CorV inactivated virus vaccine is immunologically effective. However, the duration of BBIBP-CorV-induced integrated, antibody, and T cell-mediated, immune responses needs further investigation.

[Encephalitis following COVID-19 disease]. / Covid-19 betegséget követo encephalitis.

Shortly after that COVID-19 appeared it became clear, that although the disease mainly characterized by respiratory symptoms, other signs frequently appeared, which showed involvation of other organs. There are several new publications which report about neurological complications. According to data developing of encephalitis could be relatively frequent among these. Its symptoms can mostly be observed concommittantly with respiratory symptoms or during critical state of the disease, and several forms were detected. In our patient symptoms of central nervous system involvement appeared a few weeks after healing of COVID-19 pneumonia. Clinical signs, imaging, electroencephalograpy and cerebrospinal fluid analysis confirmed the diagnosis of encephalitis. Considering the previous SARS-CoV-2 infection and the results of the examinations, we think this case was a postinfectious central nervous system disease. There are only a few data available regarding encephalitis after Covid-19 disease in the literature, yet.

A dramatic rise in serum ACE2 activity in a critically ill COVID-19 patient.

Endothelial cells express surface angiotensin-converting enzyme 2 (ACE2), the main receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that promotes the infection of endothelial cells showing activation and damage. Bronchoalveolar lavage fluid from coronavirus disease-2019 (COVID-19) subjects showed a critical imbalance in the renin-angiotensin-aldosterone system with the upregulated expression of ACE2. Recently, intravenous recombinant ACE2 was reported as an effective therapy in severe COVID-19 by blocking the viral entry to target cells. Here, we present a case of a critically ill COVID-19 patient with acute respiratory distress syndrome where circulating ACE2 was first measured to monitor disease prognosis. ACE2 activity increased about 40-fold over the normal range and showed a distinct time course as compared to 2-3-fold higher levels of endothelium biomarkers. Although the level of soluble E-selectin followed the clinical status of our patient similar to ferritin and IL-6 levels, the dramatic rise in serum ACE2 activity may act as an endogenous nonspecific protective mechanism against SARS-CoV-2 infection that preceded the recovery of our patient.

Pre-analytical practices for routine coagulation tests in European laboratories. A collaborative study from the European Organisation for External Quality Assurance Providers in Laboratory Medicine (EQALM).

Background Correct handling and storage of blood samples for coagulation tests are important to assure correct diagnosis and monitoring. The aim of this study was to assess the pre-analytical practices for routine coagulation testing in European laboratories. Methods In 2013-2014, European laboratories were invited to fill in a questionnaire addressing pre-analytical requirements regarding tube fill volume, citrate concentration, sample stability, centrifugation and storage conditions for routine coagulation testing (activated partial thromboplastin time [APTT], prothrombin time in seconds [PT-sec] and as international normalised ratio [PT-INR] and fibrinogen). Results A total of 662 laboratories from 28 different countries responded. The recommended 3.2% (105-109 mmol/L) citrate tubes are used by 74% of the laboratories. Tube fill volumes ≥90% were required by 73%-76% of the laboratories, depending upon the coagulation test and tube size. The variation in centrifugation force and duration was large (median 2500 g [10- and 90-percentiles 1500 and 4000] and 10 min [5 and 15], respectively). Large variations were also seen in the accepted storage time for different tests and sample materials, for example, for citrated blood at room temperature the accepted storage time ranged from 0.5-72 h and 0.5-189 h for PT-INR and fibrinogen, respectively. If the storage time or the tube fill requirements are not fulfilled, 72% and 84% of the respondents, respectively, would reject the samples. Conclusions There was a large variation in pre-analytical practices for routine coagulation testing in European laboratories, especially for centrifugation conditions and storage time requirements.

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy.

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

[Laboratory aspects of novel oral anticoagulant treatment]. / Az új orális antikoagulánsokkal történo kezelés laboratóriumi vonatkozásai.

The introduction of novel oral anticoagulants (NOAC) have long been expected drugs and they quickly became used widespread as their clinical effectiveness was as good as, or even better than the previously used only oral anticoagulant drug, the coumarins. Thus, the direct thrombin inhibitor dabigatran and the activated factor X inhibitors (rivaroxaban, apixaban, edoxaban) have become the part of daily therapeutic practice. Their permeation was facilitated by the guideline which suggested that no laboratory monitoring was required during NOAC treatment and this was very convenient for both patients and doctors. The clinical experience obtained in the past years, however have proved that the 'one size fits all' view is oversimplified and there are numerous situations when the determination NOAC levels is unavoidable or highly recommended. This review discusses the laboratory aspects of NOAC treatment, primarily summarizing their effect on the screening tests and special assays of hemostasis and we also describe the correct methods to determine their plasma concentrations. Orv Hetil. 2017; 158(49): 1930-1945.

Factor XIII levels and factor XIII B subunit polymorphisms in patients with venous thromboembolism.

BACKGROUND: The association of plasma factor XIII (FXIII) level with venous thromboembolism (VTE) is still controversial and the effect of sex and FXIII B subunit (FXIII-B) polymorphisms in this respect have not been explored. OBJECTIVES: 1/ To determine FXIII activity and antigen levels in patients with a history of VTE and how they are influenced by sex and FXIII-B polymorphisms. 2/ To explore the association of FXIII levels and FXIII-B polymorphisms with the risk of VTE. METHODS: 218 VTE patients and equal number of age and sex matched controls were enrolled in the study. FXIII activity was measured by ammonia release assay; FXIII-A2B2 and FXIII-B levels were determined by ELISAs. FXIII-B polymorphisms were identified by RT-PCR using melting point analysis. RESULTS: Adjusted FXIII activity and FXIII-A2B2 antigen levels were significantly higher in females with a history of VTE than in the respective controls. FXIII-B levels were significantly lower in male VTE patients than in controls. FXIII-A2B2 antigen levels in the upper tertile increased the risk of VTE in females (adjusted OR: 2.52; CI: 1.18-5.38). Elevated FXIII-B antigen level had a protective effect only in males (adjusted OR: 0.19; CI: 0.08-0.46). FXIII-B Intron K c.1952+144 C>G polymorphism significantly lowered FXIII activity, FXIII-A2B2 and FXIII-B antigen levels in both groups. FXIII-B polymorphisms did not influence the risk of VTE. CONCLUSIONS: In VTE patients the changes of FXIII level and their effect on the risk of VTE show considerable sex-specific differences. Intron K polymorphism results in decreased FXIII levels, but does not influence the risk of VTE.

Toward harmonization of interpretive commenting of common laboratory tests.

Interpretive commenting (IC) is an integral part of postanalytical activities of laboratories when the clinical interpretation of laboratory results in the context of the clinical situation of a patient is provided. Harmonizing practices in IC can be an approach to ensure high-quality comments, which if followed by adequate clinical actions has a great potential in improving patient outcomes. This paper reviews basic work prior to harmonization of IC of common laboratory test results. Practices in IC are considerably diverse both within and between countries. The quality of comments is diverse and often clinically misleading in studies that characterize and estimate error prevalence in IC. Systems that can initiate, monitor, and maintain harmonization in IC are in an evolving state. Despite international initiatives, harmonized, implementable performance indicators and goals in IC are not yet available. External quality assurance (EQA) schemes are accessible mainly in English-speaking countries. A proposal for the standard structure of EQA schemes for interpretive comments in clinical chemistry and best practice recommendations for IC are available. Few studies that demonstrate evidence on the clinical utility of IC are available in the literature. To set a strategy on further steps toward harmonization in IC, well-controlled clinical studies need to be conducted, in collaboration with laboratories and their users on the clinical usefulness of IC. Until enough evidence on the value of IC in patient outcomes accumulates, standards of qualification and training for performing IC and more EQA schemes in native languages of the users are required to improve the quality of IC.

A pragmatic approach to sample acceptance and rejection.

Sample rejection due to preanalytical errors is very common in many medical laboratories worldwide, though the decision when and how to refrain from analyzing such samples is handled very heterogeneously. As a rational, it is mostly stated that it is done to prevent the patient from being harmed by wrong medical decisions based on such values. But when thinking of the consequences of laboratory results instead of their quality being important per se, the rejection of preanalytically altered samples might be harming the patient by the need of re-collection and timely delay. The importance of the result is never the result in itself, but the consequences the result will have for the treatment or monitoring of the patient. Then again, these consequences are only foreseeable if the specific clinical situation or the general clinical setting for the respective parameter is known. Based on this mindset it can be necessary to modify general performance specifications into "personalized" performance specifications for each laboratory parameter, which can only be achieved in close interaction with clinicians. In this opinion paper we want to present a pragmatic approach to the development of such performance specifications by extending the recommendations of the 1st Strategic Conference of the EFLM on 'Defining analytical performance goals 15 years after the Stockholm Conference on Quality Specifications in Laboratory Medicine', depending on the availability of clinical information, data on biological variation or state-of-the-art recommendations, thereby redirecting laboratory medicine to a more patient focussed profession.

Regulation of plasma factor XIII levels in healthy individuals; a major impact by subunit B intron K c.1952+144 C>G polymorphism.

BACKGROUND: The regulation of plasma factor XIII (FXIII) levels in healthy individuals has been only partially explored. The identification of major non-genetic and genetic regulatory factors might provide important information on the contribution of FXIII to the risk of cardio/cerebrovascular diseases. OBJECTIVES: To determine the effect of age, smoking, BMI, fibrinogen concentration on plasma FXIII activity, complex FXIII antigen (FXIII-A2B2) and total FXIII-B subunit (tFXIII-B) level, to correlate FXIII-B level with the other two FXIII parameters and to assess the variation of FXIII levels in carriers of major FXIII subunit polymorphisms. METHODS: 268 healthy individuals were enrolled in the study. FXIII activity was measured by the ammonia release assay; FXIII-A2B2 and tFXIII-B were determined by ELISAs. FXIII-A p.Val34Leu, FXIII-B p.His95Arg and FXIII-B intron K c.1952+144 C>G polymorphisms were identified by RT-PCR using melting point analysis with fluorescence resonance energy transfer detection. RESULTS: All investigated FXIII parameters showed significant positive correlation with age and fibrinogen level; gender and BMI influenced only tFXIII-B. A highly significant positive correlation was demonstrated between tFXIII-B and the other FXIII parameters. FXIII-A p.Val34Leu polymorphism had only slight, if any effect on FXIII levels. The FXIII-B Arg95 allele moderately increased all three FXIII parameters, but the effect became statistically significant only after adjustment. The FXIII-B intron K G allele drastically decreased FXIII levels, and it seemed to be in synergism with the FXIII-A Leu34 allele. CONCLUSIONS: Plasma FXIII levels are subjected to multifactorial regulation, in which age, fibrinogen level and FXIII-B intron K polymorphism are major determinants.

Adding Value in the Postanalytical Phase.

Apart from maintaining the highest quality of analytical test results, laboratories are now getting more focused on how to achieve the greatest impact of laboratory results on their patient's outcome. Laboratory professionals are now in the learning phase of implementing new practices at different steps of the extra-analytical phases of the testing process where laboratories used to contribute seldom, only sporadically. Recently, the achievable levels of harmonization and responsible contributors at various steps of the testing process have also been proposed. Based on this proposal some tasks of the extra-analytical phase should become primarily the responsibility of laboratories with the involvement of clinicians, like additive testing, individualized interpretative commenting and reporting results with clinical urgency in postanalytical (PA) phase. These tasks can be good targets to start with or to increase patient outcome-oriented extra-analytical activities of laboratories. The status of the present practice of the PA activities for which laboratories proposed to be primarily responsible in the testing process - laboratory-driven PA tasks - will be reviewed below. In addition, approaches of quality assessment (QA) with quality specifications of these laboratory-driven PA tasks and the available best practice recommendations in the light of the achievable level of harmonization will be discussed. Laboratory professionals are encouraged to improve their methodological, theoretical and communicational skills and take the lead and participate in the discussed PA activities that can assist in translating laboratory test results into clinical meaning and thereby lead to better clinical utilization of laboratory test results.

Is D-dimer used according to clinical algorithms in the diagnostic work-up of patients with suspicion of venous thromboembolism? A study in six European countries.

INTRODUCTION: Clinical algorithms consisting of pre-test probability estimation and D-dimer testing are recommended in diagnostic work-up for suspected venous thromboembolism (VTE). The aim of this study was to explore how physicians working in emergency departments investigated patients suspected to have VTE. MATERIALS AND METHODS: A questionnaire with two case histories related to the diagnosis of suspected pulmonary embolism (PE) (Case A) and deep venous thrombosis (DVT) (Case B) were sent to physicians in six European countries. The physicians were asked to estimate pre-test probability of VTE, and indicate their clinical actions. RESULTS: In total, 487 physicians were included. Sixty percent assessed pre-test probability of PE to be high in Case A, but 7% would still request only D-dimer and 11% would exclude PE if D-dimer was negative, which could be hazardous. Besides imaging, a D-dimer test was requested by 41%, which is a "waste of resources" (extra costs and efforts, no clinical benefit). For Case B, 92% assessed pre-test probability of DVT to be low. Correctly, only D-dimer was requested by 66% of the physicians, while 26% requested imaging, alone or in addition to D-dimer, which is a "waste of resources". CONCLUSIONS: These results should encourage scientific societies to improve the dissemination and knowledge of the current recommendations for the diagnosis of VTE.

An international study of how laboratories handle and evaluate patient samples after detecting an unexpected APTT prolongation.

BACKGROUND: An unexpectedly detected prolonged activated partial thromboplastin time (APTT) can be a harmless laboratory finding, but can also reflect a thrombotic tendency or a bleeding disorder. The assistance of laboratory professionals in the interpretation of an unexpectedly detected prolonged APTT (uAPTT) is often required. The way in which uAPTTs are evaluated in laboratories was assessed in this international study with the aim of determining whether laboratory professionals are able to fulfill this need. METHODS: Postanalytical practices after uAPTT were investigated and the mixing study methodology (if used) was studied by circulating a case report with a questionnaire to staff in the invited laboratories. In addition, the interpretations of those staff regarding the presence or absence of inhibitors in three APTT mixing study scenarios were examined. RESULTS: Large within- and between-country variations were detected in both postanalytical practices and mixing study methodologies among the 990 responding laboratories, 90% of which were in 13 countries. Shortcomings regarding the investigation of uAPTTs leading to potentially incorrect or delayed clinical diagnoses were found in 88% of the laboratories. Of the laboratories to which the interpretative questions were sent, 49% interpreted all mixing study scenarios correctly. uAPTTs were investigated appropriately and all mixing study scenarios interpreted correctly in parallel in only 9.6% of the participating laboratories. CONCLUSIONS: The clinical requirement for the assistance of laboratory professionals in the interpretation of uAPTTs cannot be met at most of the participating laboratories. Laboratory professionals should be trained in the evaluation of ordinary laboratory tests, such as that for uAPTTs.

Effect of the Gas6 c.834+7G>A polymorphism and the interaction of known risk factors on AMD pathogenesis in Hungarian patients.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26-0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30-393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98-12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD.