**3. Experiments**

14 Blood Cell – An Overview of Studies in Hematology

*response* 

antigens to induce central IT.

condition. [7].

part of self.

will react with hemolysate.

absolute [2]. It is usually accompanied by a weak IR. In normal IR, one cannot identify if there is a degree of IT, because there is no defined laboratory method/test that can measure the degree of IT. Meanwhile, by logical implication, some degree of IT should exist with the

*Hypothesis I: There is no absolute immune tolerance, if and only if there is no absolute immune* 

In central IT, immature self-reactive T lymphocytes recognize antigens in the thymus and undergo negative selection (deletion) [3]. Consequently, in normal IR against a particular antigen, measuring the concentration of this antigen in the thymus can be correlated to the degree of the accompanied IT. The transport mechanism of antigens to the thymus is a critical issue because of the remarkable capacity of IS which can recognize any antigen [4]. In [5], authors claim that Dendritic Cells (DCs) have several functions, not only, in innate and adaptive immunity, but also there is increasing evidence that DCs in situ induce antigen specific unresponsiveness or tolerance in central lymphoid organs and in the periphery. The evidence that DCs transport antigens to thymus in central tolerance is very weak while the evidence that DCs have role in peripheral tolerance is more acceptable based on the review article [6]. In conclusion RBC may be vehicles which transport self

The role of RBC in transporting antigens has not been investigated before. If RBC are capable of antigen transport to induce IT, this will unveil important knowledge. For instance, in hemolytic disease of fetus and newborn (HDFN), maternal anti D alloantibody and feto-maternal ABO incompatibility are the two major causes of HDFN, Meanwhile, with the implementation of Rhesus D immunoprophylaxis, hemolytic disease due to ABO incompatibility and other alloantibodies have now emerged as major causes of this

In pregnancy, most of delivered infants are normal when there is no anti D alloantibody which means that there is an efficient mechanism that can handle the other incompatibilities. The mechanisms explained in literature explain why ABO incompatibilities, only, do not occur [8], [9] and [10], but these mechanisms do not explain why those incompatibilities occur. The mechanism may be based on trapping those antibodies in placenta through RBC catering of ABO and other incompatible blood groups antigens. Consequently, the occurrence of HDFN may be due to depletion of those antigens' store from RBC. Also, if this RBC transport function is the mechanism a body tolerates his self antigens, this will explain how a pregnant woman is able to tolerate her fetus and placenta, assuming that they are

From these *hypotheses I & II*, if RBC play role in antigen transport, one can deduce that in any mammal, blood circulating antibodies against self and foreign, either antigens or tolergens,

*Hypothesis II: RBC hide antigens to transport them to target organs.* 

normal IR. This entails that there is an equivalence relation between IT and IR.

The methodology applied will demonstrate the existence of particular self tolerogens and particular foreign antigens in RBC (Hypothesis I & II) and show that innumerable antigens exist in RBC which react with innumerable antibodies that exist in plasma. This partially proves that RBC play a role in immune reaction. To proof Hypothesis IV, it will be demonstrated that the concentration of foreign antigens in RBC varies by time in relation to IR known behavior. The experiments done are the following:


#### **3.1. Materials for experiments 1, 2, and 3**

Couples that have children, pregnant females, and single females were selected from relatives and friends. The purpose of the experiments was explained to them. Not all the combinations could be found, after blood grouping. The combinations presented, in Table-1, were used to conduct the experiments. Blood samples were taken on heparin. Some of the blood samples were used to prepare RBC and plasma and the rest was used to prepare lymphocytes using the Ficoll hypaque technique [13].

RBC were washed several times using phosphate buffer saline (PBS). The male RBC were divided into two tubes. The first tube was divided into small aliquots that were frozen to rupture RBC. The second tube was used to prepare a 5% suspension. The female RBC were divided into small aliquots that were frozen to rupture RBC. Notice that we do not need female intact-RBC.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 17

also based on competitive inhibition. Consequently, if typing wells that show positive reaction were inhibited in corresponding testing wells by adding hemolysate, this proves

the existence of specific competing antigens. Figure 2 illustrate the experiment steps.

**Figure 1.** Schematic drawing of ABO antigen transport experiment, the upper part shows how the reference positive control is conducted, while the lower part shows how the test is conducted.

**Blood**

Extract

**Lymphocytes**

**Figure 2.** Re-typing of male spouse but using his female spouse hemolysate to compete with his

Terasaki plate containing anti HLA Class I antibodies

**Hemolysat RBC**

Prepare

 A hemolysate from a third person was added to control wells. The positive control should give positive reaction. In this way, we excluded inherent errors or non-specific

First, each couple was HLA typed, and then the following was done:

Compleme

Eosin Dye

Female spouse hemolysate (diluted 1/16) was added to typing wells

lymphocytes

reaction.

**Method** 


**Table 1.** The ABO blood groups of couples used in the experiments

#### **3.2. RBC of pregnant females transport male spouse ABO antigens**

To test RBC transport of male spouse ABO antigens, a technique based on competitive inhibition of RBC agglutination was followed. If the hemolysate contains ABO specific antigens, then those antigens will compete with RBC and prevent their agglutination. Figure 1 illustrates a schematic description of the experiment.

#### **Method**

The experiment was performed, for each couple, as follows:


#### **Results**

Whenever there is ABO incompatibility and the male spouse is not 'O', agglutination was inhibited by the female spouse hemolysate and was not inhibited by male spouse hemolysate. In most cases, agglutination was inhibited in the first tube. However, agglutination was never observed in subsequent tubes. The single virgin female RBC do not contain any ABO antigens.

## **3.3. RBC of pregnant females transport male spouse HLA antigens**

This experiment was performed using commercial HLA Typing Trays for the identification and definition of HLA Class I Antigens using the microlymphocytotoxicity assay [14]. It is also based on competitive inhibition. Consequently, if typing wells that show positive reaction were inhibited in corresponding testing wells by adding hemolysate, this proves the existence of specific competing antigens. Figure 2 illustrate the experiment steps.

**Figure 1.** Schematic drawing of ABO antigen transport experiment, the upper part shows how the reference positive control is conducted, while the lower part shows how the test is conducted.

**Figure 2.** Re-typing of male spouse but using his female spouse hemolysate to compete with his lymphocytes

#### **Method**

16 Blood Cell – An Overview of Studies in Hematology

**Table 1.** The ABO blood groups of couples used in the experiments

1 illustrates a schematic description of the experiment.

her male spouse's RBC suspension.

The experiment was performed, for each couple, as follows:

**3.2. RBC of pregnant females transport male spouse ABO antigens** 

female intact-RBC.

**Method** 

**Results** 

suspension.

contain any ABO antigens.

RBC were washed several times using phosphate buffer saline (PBS). The male RBC were divided into two tubes. The first tube was divided into small aliquots that were frozen to rupture RBC. The second tube was used to prepare a 5% suspension. The female RBC were divided into small aliquots that were frozen to rupture RBC. Notice that we do not need

> Female ABO group Male ABO group O A O B O AB A O A B B A O (SINGLE) -

To test RBC transport of male spouse ABO antigens, a technique based on competitive inhibition of RBC agglutination was followed. If the hemolysate contains ABO specific antigens, then those antigens will compete with RBC and prevent their agglutination. Figure

 In positive control tubes which represent also reference tubes for comparison with test tubes, serial dilutions (up to 1/128) of female spouse plasma were made using normal saline. A drop of a male spouse's hemolysate was added before adding his RBC's

 In test tubes, serial dilutions of the female spouse's plasma were made using normal saline. A drop of the female spouse's hemolysate was added before adding a drop of

Whenever there is ABO incompatibility and the male spouse is not 'O', agglutination was inhibited by the female spouse hemolysate and was not inhibited by male spouse hemolysate. In most cases, agglutination was inhibited in the first tube. However, agglutination was never observed in subsequent tubes. The single virgin female RBC do not

This experiment was performed using commercial HLA Typing Trays for the identification and definition of HLA Class I Antigens using the microlymphocytotoxicity assay [14]. It is

**3.3. RBC of pregnant females transport male spouse HLA antigens** 

First, each couple was HLA typed, and then the following was done:


 Male spouse lymphocytes was added and followed by the complement and eosin dye as usual.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 19

washed many times with sodium citrate and then diluted with 3% formol-saline to kill any

A rabbit was injected subcutaneously with one ml of white mice RBC for four times on

The serum was examined for antibodies against mice RBC using direct agglutination

The serum was examined for antibodies against TSAs of white mice (liver, kidney and

All sections showed florescence. Figure 4 illustrates some of the histopathology sections

**Figure 4.** Histopathology sections from a white mouse's organs examined by florescent microscope showing florescence due to antigen-antibody reaction, A: kidney tissue, B: liver, and C: spleen.

Ouchterlony immuno-precipitation test of normal serum against self and other normal hemolysate was conducted, Figure 5(a). We confirmed this finding by using Western Blot technique, and showed that serum from one individual recognized antigens in hemolysate from two normal persons, Figure 5(b). Further confirmation was obtained by using twodimensional gel electrophoresis (2-DE) of co-immunoprecipitated hemolysate antigens using self-serum, Figure 5(c). Notice that the number of the immune-precipitated antigens is numerous and many spots were enriched by immune-precipitation because those antigens were not detected in 2-DE gel of hemolysate, Figure 5(d). Antigenicity of the separated proteins was confirmed by immune-blotting proteins separated by 2-DE with the same selfserum, Figure 5(e). This excluded co-precipitation of non-antigens, as they would not be

As TB is a priority disease, trying to find Mycobacterium tuberculosis bacilli protein antigens (MTPAs) in TB-patient hemolysate was conducted through 2D electrophoresis, and

**3.6. RBC hemolysate antigens are precipitated by plasma** 

bacterial contamination. An ordinary rabbit was selected to prepare the antibodies.

Blood was collected from ear-vein after 35 days from the first injection.

spleen) using the sandwich technique in histo-pathology sections.

**Method:** 

**Results:** 

weekly intervals.

taken from a white mouse's organs.

detected in immune-blotting.

**3.7. RBC transport bacterial antigens** 

slide test.

Wells that gave positive reaction in typing were examined by contrast microscope.

## **Results**

It was observed that female spouse hemolysate inhibited the typing reaction while the third person hemolysate did not. This indicates the existence of male spouse HLA antigens in female spouse hemolysate.
