Volume 35, Issue 6

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening hematological disease. An acquired mutation in the PIG-A gene in hematopoietic precursor cells leads to a deficiency of glycosylphosphatidylinositol (GPI), a common membrane anchor for certain cell surface proteins. The consequence is a deficiency of the decay accelerating factor (recognized by antibodies of the cluster CD55) and of the membrane inhibitor of reactive lysis (MIRL, recognized by CD59 antibodies) on the surface of blood cells leading to dysregulation of the complement cascade resulting in characteristic intravascular hemolysis and thromboembolic complications. The analysis of GPI-anchored proteins on blood cells is the recognized diagnostic gold standard. Some aspects of the recently published international guidelines are still a matter of discussion. On the basis of these guidelines, we discussed practical recommendations which led to the German-Austrian consensus paper presented here. It contains specific proposals for indication of the test, preanalytical aspects, how to perform immunophenotyping and for interpretation of results. We propose a two-step diagnostic approach. Step 1 is a screening and step 2 is a confirmation of the diagnosis. The latter should be restricted to laboratories who have substantial experience with PNH. According to this algorithm for a stepwise diagnostic approach we recommend which antibody panel should be used in which step. The most frequent sources of error and how to avoid them are also summarized. This consensus should support medical laboratories in making an early and correct diagnosis of what is important to the therapy and prognosis of the patient. Aspects of economic efficiency and feasibility were also included in our considerations.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening hematological disease. An acquired mutation in the PIG-A gene in hematopoietic precursor cells leads to a deficiency of glycosylphosphatidylinositol (GPI), a common membrane anchor for certain cell surface proteins. The consequence is a deficiency of the decay-accelerating factor (recognized by antibodies of the cluster CD55) and of the membrane inhibitor of reactive lysis (MIRL, recognized by CD59 antibodies) on the surface of blood cells leading to dysregulation of the complement cascade resulting in characteristic intravascular hemolysis and thromboembolic complications. The analysis of GPI-anchored proteins on blood cells is the recognized diagnostic gold standard. Some aspects of the recently published international guidelines are still a matter of discussion. On the basis of these guidelines, we discussed practical recommendations, which led to the German-Austrian consensus paper presented here. It contains specific proposals for indication of the test, preanalytical aspects, how to perform immunophenotyping, and for result interpretation. We propose a two-step diagnostic approach. Step 1 is a screening and step 2 is a confirmation of the diagnosis. The latter should be restricted to expert laboratories’ experience with PNH. According to this algorithm for a stepwise diagnostic approach, we recommend which antibody panel should be used in which step. The most frequent sources of error and how to avoid them are also summarized. This consensus should support medical laboratories to make an early and correct diagnosis of what is important to therapy and prognosis of the patient. Aspects of economic efficiency and feasibility were also included in our considerations.

Recently, several novel autoantibodies have been identified which are closely associated with different subtypes of autoimmune encephalitis. These antibodies are directed against structures located on the neuronal cell surface: glutamate receptors (types NMDA and AMPA), GABA B receptors, as well as the voltage-gated potassium channel-associated proteins LGI1 and CASPR2. They are much more common than the classical paraneoplastic antibodies (anti-Hu, -Yo, -Ri, -Ma, -CV2, -amphiphysin), less frequently associated with a tumour, and the corresponding clinical syndromes respond significantly better to immunotherapy. Monospecific detection of these autoantibodies in the serum or cerebrospinal fluid of patients is primarily performed by indirect immunofluorescence using transfected HEK293 cell lines recombinantly expressing the membrane-associated target antigens. Owing to the symptom overlap of the respective disorders, it is highly appropriate to determine these parameters in parallel for each patient (autoantibody profiles). Early diagnosis (substantially supported by the serological laboratory), the immediate initiation of immunotherapeutic intervention and, in cases of paraneoplastic aetiology, tumour resection are crucial for prognosis. In our own investigations, antibodies against glutamate receptors (type NMDA) are most frequently found among the newly identified forms of autoimmune encephalitis, accounting for 42% of cases. In laboratory practice, one-third of positive reactions were caused by an autoantibody whose determination was not requested by the clinician. Considering the clinical significance of a positive result, these findings substantiate the need to implement multiparametric serological test systems in this diagnostic area.

Recently, several novel autoantibodies have been identified which are closely associated with different subtypes of autoimmune encephalitis. These antibodies are directed against structures located on the neuronal cell surface: glutamate receptors (types NMDA and AMPA), GABA B receptors, as well as the voltage-gated potassium channel-associated proteins LGI1 and CASPR2. They are much more common than the classical paraneoplastic antibodies (anti-Hu, -Yo, -Ri, -Ma, -CV2, -amphiphysin), less frequently associated with a tumor, and the corresponding clinical syndromes respond significantly better to immunotherapy. Monospecific detection of these autoantibodies in the serum or cerebrospinal fluid of patients is primarily performed by indirect immunofluorescence using transfected HEK293 cell lines recombinantly expressing the membrane-associated target antigens. Owing to the symptom overlap of the respective disorders, it is highly appropriate to determine these parameters in parallel for each patient (autoantibody profiles). Early diagnosis (substantially supported by the serological laboratory), the immediate initiation of immunotherapeutic intervention and, in cases of paraneoplastic etiology, tumor resection are crucial for prognosis. In our own investigations, antibodies against glutamate receptors (type NMDA) are most frequently found among the newly identified forms of autoimmune encephalitis, accounting for 42% of cases. In laboratory practice, one-third of positive reactions were caused by an autoantibody whose determination was not requested by the clinician. Considering the urgency for therapeutic measures in positive cases, these findings substantiate the need to implement multiparametric serological test systems in this diagnostic area.

The role of procalcitonin (PCT) plasma levels as a diagnostic tool for intensive care patients has been intensively investigated during the past years. In particular for recognition of bacterial infections, PCT levels have been shown to be superior to other clinical and biochemical markers. Furthermore, some very recent studies show that in patients with lower respiratory tract infections PCT guided antibiotic therapy reduces antibiotic use and thereby may also reduce duration of stay of patients in hospital and thus cut hospitalisation costs. However, various studies indicate that the value of PCT as a prognostic marker is limited because of false positive or negative values. Despite these limitations PCT plasma levels are currently measured in intensive care units. The present study summarises the possible clinical uses of this lab marker as a diagnostic tool for the assessment of critically of ill patients.

The role of procalcitonin (PCT) plasma levels as a diagnostic tool for intensive care patients has been intensively investigated during the past years. In particular for recognition of bacterial infections, PCT levels have been shown to be superior to other clinical and biochemical markers. Furthermore, some very recent studies show that in patients with lower respiratory tract infections PCT guided antibiotic therapy reduces antibiotic use and thereby may also reduce duration of stay of patients in hospital and thus cut hospitalisation costs. However, various studies indicate that the value of PCT as a prognostic marker is limited because of false positive or negative values. Despite these limitations PCT plasma levels are currently measured in intensive care units. The present study summarises the possible clinical uses of this laboratory marker as a diagnostic tool for the assessment of critically ill patients.

Analysis of autoantibodies (AABs) by indirect immunoflu­orescence (IIF) remains the hallmark of diagnosing autoimmune diseases despite the introduction of multiplex techniques. Non-organ specific AABs are screened in routine diagnostics by IIF on HEp-2 cells. However, IIF results vary due to objective (e.g., cell fixation) and subjective factors (e.g., expert knowledge). Therefore, inter- and intralaboratory variance is relatively high. Standardisation of AAB testing by IIF remains a crucial issue in and between routine laboratories and may be improved by automated interpretation systems. A novel automated reading system provides fully automated reading of fluorescence images on HEp-2 cells and software algorithms for the mathematical description of AAB staining patterns. It can be used for screening and preclassification of non-organ specific AABs in routine diagnostics regarding systemic autoimmune and autoimmune liver diseases. More sophisticated pattern recognition algorithms and novel calibration systems will improve standardised quantifications of IIF image interpretation.

Autoantibodies (AAb) with high diagnostic specificity are important markers for diagnostics and differential diagnostics of autoimmune diseases (AIDs). A negative result of certain AAb or AAb combinations may exclude the appropriate AID. By contrast, AAb may lead to a definite diagnosis even if the disease might not be diagnosed according to the diagnostic or classification criteria. Because optimal control of disease development requires early and adequate therapy, the diagnosis must be definite at the earliest possible stage of the disease. Therefore, determination of AAb with high diagnostic specificity and high predictive values become more and more important for early and predictive diagnosis of AIDs.

The for the most part long preclinical stages, the large variability of clinical manifestations as well as the need of an early adequate therapy of autoimmune diseases require the search for and evaluation of applicable biomarkers, which reliably reflect the disease process and allow for a definite early diagnosis as well as for therapeutically relevant evidence (disease activity and course, organ manifestations, reliability of different types of therapy). To date, mainly autoantibodies have been used for serological diagnostics, prognostics and partially also for monitoring of autoimmune diseases. Dependent on the diagnostic variables they are important diagnostic markers but the usefulness for prognostics, theranostics and disease monitoring is rather low. Therefore, the purpose of future developments should be to find adequate combinations of disease relevant autoantibodies and other biomarkers to realize a reliable early diagnosis as well as to obtain useful information regarding clinical course and reliability of different types of therapy. Such multiparametric analyses are the prerequisite for an optimized personalized therapy and therefore for the improvement of the medical care of patients with autoimmune diseases.