Semi-Solid Phase Assay for the Alternative Complement Pathway Activity Assessment (AP 100 )

Since the introduction of the most expensive drug in the world (Eculizumab) in the therapeutic arsenal of many diseases involving the alternative complement pathway (ACP) in their pathophysiology, the unmet need to perform simple ACP assays affordable for all countries has become one of the major challenges of the contemporary medicine. The assay currently used is AH 50 , despite it still challenging for several laboratories. This educational chapter consists on a detail protocol of standardized hemolytic assay AP 100 and aims to help clinical laboratories over the world and especially those of the developing and low incomes countries to perform it. The procedure is essentially the same as for the timed lysis assay and dilution methods (AP 50 ) except the concentration of ACP buffer and the chicken erythrocyte density used to make the gels. In clinical field, AP 100 has at least nine applications in disease diagnosis and follow-up. AP 100 has many advantages over the AH 50 as it is more reliable for the Eculizumab monitoring and more practical with a purpose to be stored and transported for several weeks. AP 100 is a portable and easy to use device both at the bedside and in the companion medical care.


Introduction
Complement system is the pillar of the immune system by its dual role in homeostasis and disease. It is the first line of the innate immunity and augments adaptive immunity. Indeed, complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders through a complex cascade of proteases [1]. Activation of the pathway occurs through three primary pathways: classical, lectin, and alternative pathways. Instead to the other pathways and in addition to properdin as the initiating molecule, the alternative complement pathway (ACP) is activated via a low level of constitutive spontaneous hydrolysis of C3 in a process known as tick-over. Importantly, thanks to its amplification loop, ACP plays a major role for the final effect of initial specific activation of the classical and lectin complement pathways and contributed to 80-90% of any C5 activation regardless the initiating pathway [2]. Interestingly, ACP has been shown to play a particularly important role in preclinical disease models [3].
The ACP functional assessment constitutes an unmet need in medicine and applied research fields as the health valorization of bio-molecules extracted from nature. In clinical field it has at least nine applications in disease diagnosis and follow-up. For instance in therapy monitoring, it allows to screen patients responders to the complement blockers like Eculizumab, a patient with abolished activity means that he has no C5 mutation and is considered as eligible to this therapy. Furthermore, ACP activity makes possible to assess drug effectiveness at the plasmatic level [4]. Moreover, by evaluating ACP function we can predict and avoid immune-complex diseases flares and end organs damages as in systemic lupus erythematosus (SLE).
For the disease diagnosis, several international consensuses include functional hypocomplementemia and ACP abnormalities as a diagnostic criterion: 1. Kidney diseases resulting from abnormal control of ACP especially atypical hemolytic uremic syndrome (aHUS), C3 glomerulonephritis (C3GN), and densedeposit disease (DDD), as well as atypical postinfectious glomerulonephritis [5].
2. Hypocomplementemia by a hemolytic assay constitutes one point in the diagnosis score of EULAR/ACR Lupus Classification Criteria 2017 and useful marker for evaluating SLE renal disease activity and outcomes [6].
3. The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is the chronic intravascular hemolysis that is a consequence of unregulated activation of ACP [7].
4. Individuals deficient in components of the alternative and terminal complement pathways are highly predisposed to invasive, often recurrent meningococcal infections [8]. The most frequent bacterial meningitides related to complement proteins deficiencies are dues to factor B, factor D and membrane complex attack proteins deficiency.  7. ACP is particularly considered in sepsis, due to its uncontrolled amplification in sepsis conditions [10].

Protocol
The procedure is essentially the same as for the ACP by kinetic fluid phase assay except the concentration of AP buffer and the chicken erythrocyte density used to make the gels [11]. A value of 100% of plasma ACP function should be defined by the pooled normal human plasmas (NHP standard), prepared from a total of 100 healthy individuals separate for each sex.

Samples
Patient serum samples for the functional hemolytic assays need to be fresh, that is serum should be separated on the day of venepuncture and used the same day, or stored at −80°C. This is probably the single most difficult, yet important, step because if the cold chain is broken, the results become impossible to interpret correctly [12]. Whole blood, with ethylenediaminetetraacetic acid (EDTA) as the anticoagulant can also be used.
Chicken red blood cells (CRBC) was collected in tubes containing Alsever or 20% (v/v) acid citrate dextrose (ACD) and stored at 4°C.  [13]. This prevents complement activation via the classical pathway and facilitates complement activation via the ACP in agarose gels. ACP buffer/gelatin contains 1 g/L gelatin and is used when protein concentrations are low.

Buffers and other reagents
3. Agarose: use an agarose that has a low melting point as plates are easier to pour.

Procedure
1. Before starting, in a sterile tube put 9.8 mL of 1×ACP buffer at 56°C. Also let warming some Petri dish (or rectangular plate) in the 56°C incubator.

Representative results
After incubation, the ACP activity is calculated after measuring the diameter using the vernier caliper or taking the ACP dish in photo and measure each surface well by Image J® (Figure 1). Area surfaces are calculated by the formula S = π*r 2 , Semi-Solid Phase Assay for the Alternative Complement Pathway Activity Assessment (AP 100 ) DOI: http://dx.doi.org /10.5772/intechopen.81743 with r = d/2. (r: radius, d: diameter, s: surface). The same procedure is applied to the different NHP dilutions included to establish the calibration curve. This is because diluting NHP means that all ACP components are diluted equally. As example of interpretation, the more the ring is small the more the effectiveness of Eculizumab is good. The absence of reaction in a well signifies a complete ACP blockage, either by a drug or by a pathological process.
6. N-saline is 9 g NaCl dissolved in 1 L H 2 O.
7. Barbitone buffer is made by mixing 0.1 M solutions of sodium barbitone and barbituric acid to obtain the target pH and adjusting volume to obtain required final molarity.

Equipment
1. Suitable Petri dishes or glass plates. Size depends on the number of samples. Volumes can be adjusted so that the final depth of the gel is ~1-1.5 mm.
2. 56°C incubator and dishes/plate warmer (water bath can also be used).
6. Pasteur pipette as well cutters to produce holes approximately 5 mm diameter. If single holes are to be punched, a grid should be placed under the gel so that the holes can be distributed evenly.

Discussion
Since Thomas A. E. Platts-Mills and Kimishige Ishizaka have discovered that fresh normal human serum in EGTA buffer was found to cause >90% hemolysis of unsensitized rabbit red blood cells (RaRBC) [11], simple timed lysis assay and dilution methods called AP 50 (Alternative Pathway 50) was performed to quantify hemolytic complement activity in human serum [13][14][15]. This reaction requires C3, factors B and D, and Mg ++ ions to form the C3 convertase (C3bBb) [14,15]. In AP 50 , ACP is activated and measured by virtue of RaRBC decreased sialic acid content in addition to the blockade of the classical pathway activation by chelation of calcium by EGTA [16]. Nonetheless, AH 50 requires a lot of material like spectrophotometer and consumable test tubes and highly skillful personal. To adapt this assay to simple labs, a semi-solid phase assay was proposed to measure ACP activity by using chicken erythrocytes incorporated in agarose gel called AP 100 . Cell membranes of chicken erythrocytes have the same properties as those of the rabbit in terms of cell surface charge density of sialic acid whereas they are more robust than rabbit because they have nuclei. AP 100 assay has many advantages over the AH 50 one ( Table 2). For example, AP 100 is more reliable for the eculizumab monitoring as shown in the article we have recently reviewed [17]. Moreover, AH 50 still challenging in low incomes country laboratories that cannot equip their hospital and research laboratories to perform this assay. To overcome this roadblock, it was proceeded to render this method more practical to each laboratory with a purpose to be stored and transported for several weeks. This includes a hemolytic agarose dishes/plates; a special adaptation to prevent complement activation via the classical pathway and facilitate complement activation via the alternative one in agarose gels.
To the best of our knowledge there were no laboratory performing that assays in developing countries especially in Africa and no educational chapter is available to explain it. Once its optimization in each of these countries laboratories was performed, thanks to ACP dishes/plates, we are expecting to empower doctors' decision making process and improve quality of patients' management and therapy follow-up. Therefore this portable and easy to use device even at the bedside of the patient and in the companion do not necessitate any equipped laboratory and may facilitate prospective analysis and disease screening in large populations. With enlargement of the ACP disease spectrum necessitated complement blockade, AP 100 should be considered by clinical laboratory scientist especially to analyze sets of samples at once.