**5. How this RBC antigens transport function can be exploited**

This observed RBC antigens transport function creates an antigens' store. This store can be exploited in many directions. The proposed direction is to exploit functional proteomics approach [16] with the following three crucial aspects of the experimental design to produce products which are among diagnostic kits, vaccines or treatment components:


The aim of this approach is to identify antigens which are relevant to a particular disorder.

#### **5.1. Direct approach for products development**

This approach is based on using a subset of antibodies which are specific against a subset of antigens of a particular disease to enable the use of those antibodies and those antigens in preparation of beneficial products.

Diagnostic kits can be prepared for all infectious microorganism and all tumors. In such disorders, simple kits can be prepared using the following steps:


A more advanced kits based on selection of antigen-determinant sites (epitopes) can be prepared. The problem of such kits, which uses a particular antigen, is in its validation which will be more sophisticated. One can expect that this particular antigen may not exist in RBC antigens' store of some population who are genetically different from the population used in preparation of the kit.

Active vaccines against all infectious microorganism and all tumors can be prepared by using the purified antibodies prepared for diagnostic kits in identifying related antigens existing in RBC antigens' store. The identified antigens can be prepared using the technology of recombinant proteins purification.

#### **5.2. Bioinformatics approach for products development**

The proposed mathematical model and a data mining algorithm will not only help in identifying proteins (antigens) that can be used in diagnosis and treatment of difficult disorders, but also will help in etiological diagnosis of idiopathic disorders and their treatment. This approach is based on building large databases of RBC antigens' store for patients and normal individuals. Consequently, a patient sample is collected on anticoagulant. RBC and plasma are separated. The plasma IgG is separated and then used as ligand in immunoaffinity chromatography to separate hemolysate antigens. The collected antigens are identified by mass spectrometry. The database record consists of the diagnosis and the set of identified antigens.

#### *5.2.1. Mathematical model*

It consists of four main parts; definitions of symbols, model of diseases caused by microorganisms, tumors, or foreign proteins; model of autoimmune diseases which result as a consequence of missed tissue proteins from RBC antigens' store; and model of diseases of unknown cause (Idiopathic).

#### **Definitions**

24 Blood Cell – An Overview of Studies in Hematology

analyzed by mass spectrometry

**5.1. Direct approach for products development** 

disorders, simple kits can be prepared using the following steps:

2. Prepare hyper immune serum using extracted antigens

related antibodies from hyper immune serum

5. Adsorb purified antibodies to latex beads

technology of recombinant proteins purification.

banks available

preparation of beneficial products.

3. Build an affinity column

used in preparation of the kit.

disorder.

tolerance to that antigen will eventually vanish. Consequently, an autoimmune response

This observed RBC antigens transport function creates an antigens' store. This store can be exploited in many directions. The proposed direction is to exploit functional proteomics approach [16] with the following three crucial aspects of the experimental design to produce

1. The strategy used for the selection, purification and preparation of the antigens to be

3. The method used for the interpretation of the mass spectrometry data and the search engine used for the identification of the proteins in the different types of sequence data

The aim of this approach is to identify antigens which are relevant to a particular

This approach is based on using a subset of antibodies which are specific against a subset of antigens of a particular disease to enable the use of those antibodies and those antigens in

Diagnostic kits can be prepared for all infectious microorganism and all tumors. In such

4. Antibodies purification: Use affinity column containing antigens to separate their

A more advanced kits based on selection of antigen-determinant sites (epitopes) can be prepared. The problem of such kits, which uses a particular antigen, is in its validation which will be more sophisticated. One can expect that this particular antigen may not exist in RBC antigens' store of some population who are genetically different from the population

Active vaccines against all infectious microorganism and all tumors can be prepared by using the purified antibodies prepared for diagnostic kits in identifying related antigens existing in RBC antigens' store. The identified antigens can be prepared using the

1. Extract antigens from microorganism/tumor-cell-line cultures in coupling buffer

will be provoked to that antigen and autoimmune disease is established.

**5. How this RBC antigens transport function can be exploited** 

products which are among diagnostic kits, vaccines or treatment components:

2. The type of mass spectrometer used and the type of data to be obtained from it

Let the assumption of this work be as the following:

pi: protein amino acid sequence, where i = 1 .. n

dj: health state, i.e., normal or disease name, where j = 1 .. m

P = {p1, …, pn}, Set of all proteins of RBC antigens' store

D = {d1, …, dm}, Set of all diseases

Pp: patient proteins where Pp P where p is the patient ID

Op: (pi , dj), ordered pair of patient presented by protein sequence (i) and health state (j).

a. Model of Diseases caused by microorganisms, tumors, or foreign proteins

$$\mathbf{P}\_{\mathrm{dj}} = \cap \{ \mathbf{P}\_{\mathrm{P}} \}\_{\mathrm{dj}}$$

Where Pdj is the set which contains all common proteins associated with dj.

$$\mathbf{P}\_{\mathrm{normal}} = \cup \{ \mathbf{P}\_{\mathrm{P}} \}\_{\mathrm{normal}}$$

Where Pnormal is the set which contains proteins associated with normal.

P'normal such that p in Pnormal if the number of occurrence of p Pnormal is less than 5% of the total number of p in Pnormal then remove p from Pnormal.

$$\mathbf{P'}\_{\mathrm{dj}} = \mathbf{P}\_{\mathrm{dj}} - \mathbf{P'}\_{\mathrm{normal}}$$

Where P'dj is the set which contains proteins that can be used as biomarker or vaccines, Figure 10.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 27

Applying data mining methods (A and B) can help to identify new diseases and treat

Patients' blood samples will be collected on anticoagulant. RBC and plasma are then separated in different tubes. Plasma is used as a ligand in immune-affinity chromatography to separate hemolysate antigens that can bind to plasma antibodies. The separated antigens are identified by MS and stored in the database indexed by the patient

In the same time, queries are done to verify the diagnosis and get a prognosis and a recommended treatment component. The following formulas describe the usage of this

∀ P'dj ∈ Dp', if P'dj ⊂ P, then patient is diagnosed to have dj

P''dj ∈ Dp'', if P''dj P, then patient is diagnosed to have dj

All the previous work in RBC proteomics neither has identified another function nor has mentioned the finding of: HLA, TSAs, or foreign proteins. The reasons are obvious. Firstly, it is not expected to find such proteins and consequently the method used for the interpretation of the mass spectrometry data, and the search engines used for the identification do not consider the right types of sequence data banks available. Secondly, the amount of most of the antigens which belong to the RBC antigens' store is little. This makes

The work described is just a pilot study that throws some light on a new theory related to RBC. This theory is based on finding antigens' store consisting of self and non-self antigens. Although this theory can be related to immune tolerance by logical induction, the concrete evidence and mechanism need further research. Mainly, the logical induction is based on finding all kind of antigens in hemolysate, especially HLA antigens which are related to fetus. This existence of all kinds of antigens, definitely, plays some immunological role

The initial experiment, which shows the existence of ABO antigens in hemolysate of pregnant females, explains the mechanism of how HDFN occurs. Meanwhile, the

patients appropriately.

disorder.

Then

Else

**6. Conclusions** 

model in clinical practice.

Let Dp' is the set of all discovered P'dj Let Dp'' is the set of all discovered P''dj

those antigens invisible and hence easily missed.

which may be immune tolerance.

*5.2.2. Scenario of the system in clinical environment* 

**Figure 10.** Venn diagram depicting set of abnormal protein of disease X (P'dj)

b. Model of Diseases caused as a result of missed tissue proteins

Pudj = ∪{Pp}dj

The result is the set which contains all proteins associated with dj

$$\mathbf{P}^{\ast \ast} \mathbf{d}\_{\mathrm{d} \mathfrak{j}} = \mathbf{P}\_{\mathrm{normal}} \mathbf{-P}^{\mathbf{u}} \mathbf{d}\_{\mathrm{d} \mathfrak{j}}$$

The result is the set which contains proteins that can be used to diagnose patients through detecting circulating auto-antibodies and to treat those patients through desensitizing them with the proteins that give positive reaction, Figure 11.

**Figure 11.** Venn diagram depicting set of missed normal proteins of disease Y (P''dj)


Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 27

 Applying data mining methods (A and B) can help to identify new diseases and treat patients appropriately.
