**2. Protocol**

*Biochemical Testing - Clinical correlation and Diagnosis*

Immune complex (IC) diseases, SLE with and

Mesangiocapillary glomerulonephritis (MCGN) or membranoproliferative glomerulonephritis

Occasionally disseminated *N. gonorrhea* infections.

*Clinical presentations of ACP proteins acquired or genetic abnormalities.*

Severe, occasionally repeated, *N. meningitidis*

Neisserial infections, usually recurrent

meningococcal infections.

infections in males

without glomerulonephritis (GN).

(MPGN)

**Table 1.**

**Disease presentation ACP proteins involved**

Most of ACP proteins trigger tissue injury because ACP constitutes the amplification loop of the

fH or fI deficiency, or the presence of C3 nephritic factors. Can also occur with classical component

C3 deficiency classically caused by C3 nephritic

Terminal component (C5–8) deficiencies; the association is less strong in patients with C9 deficiency. Patients are usually otherwise healthy.

Properdin deficiency or Factor D deficiency (very

which lack glycosylphosphatidylinositol (GPI) membrane anchored Complement control proteins

classical pathway

factors, also fH deficiency

rare, not sex linked)

[(DAF) And CD59].

deficiency.

Severe and often repeated pyogenic infections. Deficiency of C3 either primary, or secondary to

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

Paroxysmal nocturnal hemoglobinuria Rare acquired clonal disorder of hemopoietic cells

For the disease diagnosis, several international consensuses include functional

hemolytic uremic syndrome (aHUS), C3 glomerulonephritis (C3GN), and densedeposit disease (DDD), as well as atypical postinfectious glomerulonephritis [5].

1.Kidney diseases resulting from abnormal control of ACP especially atypical

2.Hypocomplementemia by a hemolytic assay constitutes one point in the diagnosis score of EULAR/ACR Lupus Classification Criteria 2017 and useful

3.The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is the chronic intravascular hemolysis that is a consequence of unregulated activa-

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

marker for evaluating SLE renal disease activity and outcomes [6].

hypocomplementemia and ACP abnormalities as a diagnostic criterion:

**104**

erythematosus (SLE).

tion of ACP [7].

complex attack proteins deficiency.

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.

## **2.1 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.

#### **2.2 Buffers and other reagents**


#### **2.3 Procedure**


Transfer one bottle with diluted agarose to 45°C and allow to cool to this tempe rature.

