()

(Immunohematology) :-

.

Immunohematology :-Is defined as the study of the immunology of blood and includes those principles and practices that are known collectively as blood banking.

( ) :- . () . :- . :-1- .2- :- - . - (Rhesus factor) . . (identification of irregular Abs).- .3- .4- .5- .6- .- :- :-

1- .

2- .

3- .

4- :-

- () .

2- ( ) .

- .

8- .

- (filtration) () .

27- ( ) .

22- * .

* .

:-

1- () .

2- .

- .

8- .

(Blood Donor Selection) :- . . :- 1. .

2. .

3. .

: :-1. ( ) .

2. ( ) . :- :-1- :- 18 65 45 65 .

2- :- 47 50 ( 12 13 % ) 495 11 / .

3- Hb 135 / 125 / Hct 0.38 38 % 0.39 39 % .

4- :- 12 ( 4 ) 16 .

5- 80 / 100/ .

Systolic pressure should be no greater than 180 mm Hg, and the diastolic pressure should be no greater than 100 mm Hg.

5 10 .

6- :- 50 100 .

7- :- 37.5 .

8- .

:- :-1- .

2- 6 .

3- (German measles) ( yellow fever) (small pox) (mumps) ( ) (Rabies vaccine) . (HBV) B 6 . B (Hepatitis B immunoglobulin) . ( ) () (Passive immunization) .

4- 6 .

5- (yellow fever) (Relapsing fever) .

6- .

7- .

8- ( ) .

9- .

10- 72 48 . :-1- B C . .

2- .

3- .

4- (pretransfusion reactions) .

5- .

6- .

7- (Leukemia) .

8- . :-1- .

2- .

3- . * . Medical History :-

. .1. (Questions to protect the donor) .

2. (Questions to protect the recipient) . :-1- . . .

2- . (angina). .

3- ( TB) . (TB) .

4- (Blood disease) (Abnormal bleeding) (clotting problems) . (lymphomas) . A B (Hemophilia A & B) .

5- () (Convulsions) (seizures) (fainting) (epilepsy) . .

6- . ( ) .

7- (prescription) ( ) (over-the-counter medication) . :-- Accutane 30 .- Retine-A ointment .- Tegison 3 .- Aspirin 3 .- .- (anti-malarial drug) 3 .- (Human growth hormone) ._ (Marijuana & Alcohol) .8- 6 . . : :-1- (yellow jaundice) ( ) (tattoo) ( ) . . B C (cirrhosis of the liver) .

2- . 3 .

3- (HIV1 & HIV2 +ve) .

4- . .

5- . .

6- ( ) .

7- .

Care of Donor :- ( ) .- . ( ) :-- . - .- 30 . .- . - 8 . - . - . - 3 ( ) .* .

( ) Donor Reactions :- :1- () (Grade I Reactions) :-

(psychological factors) (emotional stress) (paleness) (weakness) (perspiration) () () .* .2- (Grade II Reactions) :- (fainting) (hypotension) (convulsion or tetany) .* () (tetany).

3- (Grade III Reactions):- (dilated and fixed pupils) (snoring) . .

() (Hemaphersis) :-

.

.

( ) :- (multiple bags) (Refrigerated centrifuge) .

. 450 ( ) . (satellite big) saline .

:- () :

1. (Continuous flow centrifugation) .

2. (Intermittent flow centrifugation) .

3. (Filtration membrane) : .

1- (Continuous flow or) - (An intermittent flow) .

.

. .

.

(one venipucture) .

(Whole Blood) :-

450 63 CPDA1 CPD .

- ( ) 25% . haematocrite 1.5% 24 () (Labile coagulation factors) (factor VIII) (factor V) .

35 CPD CPDA1 1 6 42 () (Additive solution) SAG-M 42 .

.

:

CPDA (CPDA Whole Blood)

(Collection time and date) : (Expire date) :- .

(ABO group) : .

(RBCs) :-

. (Fluid overload) . (To increase RBCs mass) (Hb) (haematocrit) 0.03 (3%) 1.5% .

( 1 6 ) .

24 35 CPDA1 CPD ( ) (additive system solution) (additive solution) saline Adenine Glucose Mannitol (SAG-M) . 42 1 6 .

(Washed RBCs) :-

(Saline solution) () (debris) .

COBE2991 . (saline) (Centrifugation) (supernatant) . (hematocrite) 60 80% .

(saline) 90 % 98 % 95 % .

5 x 810 80 % ( ) .

() 24 1 6 .

:-

1 -A (anti-A) .

2 (Paroxysmal Nocturnal Haemoglobinuria = PNH) .

3 .

4 .

(Red Cell Concentration) :-

(centrifugation) (Sedimentation) .

()

(RBC concentrated in optimal additive solution) :-

(additive solution) SAG-M :

1- Sodium Chloride 140 mmol/l.

2- Adenine 1.5 mmol/l.3- Glucose 50 mmol/l

4- Mannitol 30 mmol/l. 28 35 . 0.50 70 .0 ( ) . (neonates) .

(Filtered RBC) :-

.

(Leukocytes-reduced RBC) :-

80 % 5 X 810 (HLA)

(CMV) 5 X 10 .

(HLA) (Patients who are already sensitized to HLA () (febrile reaction) () .

(CMV) (EBV) . ( ) (Cell-associated Virus) .

. .

(Rejuvenation) :-

2,3,DPG ATP (shelf life) 37 Pyruvate, inosine, Glucose and Phosphate () (quality) .

(Rejuvenation) ( 1 6 ) . 24 . () .

Frozen/Thawed, Deglycerolized RBC :-

(Dehydration) (Cryoprotetive agent) . .

( ) :-

1. .

2. .

3. ( ) (CMV) (Rubella) .

4. 0) (Hemolytic transfusion reaction) .

5. .

6. ATP 2,3,DPG .

7. .

40 47% (6.2M) 65 (65 C) 14 17% 120 (- 120 ) .

(Deglycerolization):-

(thawing) 37 () 37 . (hypertonic solution) (12% ) (normal saline) 0.2% .

24 .

:-

1. .

2. 500 .

3. 200 / (Free plasma Hb not less than 200 mg /l) .

(Filtered RBCs) :-

223 .

Buffy Coat Transfusion:-

buffy coat (anti-microbial therapy) 9 . buffy coat .

buffy coat (human granulocyt-colony stimulating factor) buffy coat .

(Plasmaphersis) :-

Is defined as collection of plasma by withdrawal of whole blood followed by the separation of plasma and reinfusion of the cells. The plasma is used similarly to fresh frozen plasma. The volume of plasma collected is normally 600 ml.

(Fresh Frozen plasma = FFP) :-

6 . 12 30 (-30c) . (immunoglobulin) C1-esterase inhibitor .

( ) .

(Blood Bank Plasma) :-

1 6 6 . .

(Lyophilized Plasma) :-

(Dry Freezer) 1 6 . .

(Plasma Derivatives) :-

. :-

1. (Albumin solution) :-

4.5% 20% . (hypoproteinaemia) (hypovolaemia) (pancreatitis) .

.

96% 4% .

Plasma Protein Fraction: -

This contains approximately 83% albumin and 17% globulins. (Cryoprecipitate) :-

(cryoprecipitate is the cold, insoluble fraction of plasma) 1 6 :-

1. Fibrinogen 250 mg. .

2. Factor XIII, Fibrinoctin, and the procoagulant and Von willebrand portion of factors VIII .

80 120 (factor VIII) .

:-

1. (haemophilia A) .

2. (Von Willebrand disease).

3. () (congenital or acquired fibrinogen deficiency) .

4. 13 (Factor XIII deficiency) .

5. (Some platelet function disorders) .

18 6 18 4 .

.

:-

1- (Factor VIII concentrates) .

2- (Factor IX concentrates) .

(factor II) (factor X) () . VII XI XIII activated prothrombin (anti-thrombin III) .

:-

:

1. ( ) :- :

1- RhD immunoglobulin () 72 .

2- Zoster Immunoglobulin : Herpes viricella zoster infection .

3- Standard human immunoglobulin :- ( ) .

3- Hepatitis Diphtheria Tetanus Rubella Rabies Measles CMV Pseudomonous infections .

2. (Immunoglobulin for intravenous use) :-

. ( ) () auto-immune thrombocytopenia . : : ( ) . ( ) . ( ) (Pilot Sample) 30 . . 5 :- .- .- .- .- : / .- . ( ) . 450 ( 45 63 CPD CPDA1 570 (trip balance) (counterweight) 8 10 .

. . 30 (segments sample) . .

10 15 () . .

50 450 (45 ) 30 . 50 3750 405 495 11% 13% . (hypovolemia) 15% . ( 50 ) . :1.

2. 300

: :

( 50) X 450 =

:

:- () 450 (45 ) 50 . 300 .

CPD CPDA-1 14 100 :-

= ( 100) X 14

. :-

= 63

(satellite bag) 1 . .:

30 (whole blood) .:

1- : (30 50) X 450 = 270 .2- : (270 100) X 14 = 37.8 .3- : 63 37.8 = 25.2 . : :-

1. 1 6. 1 10.2. 30.3. 65 -196. 65 85 150 196 . . . :-

The Purpose of Anticoagulants and Preservative Solutions

1. .

2. .

3. .

() (Production of Blood Bank Reagent) :-

(screening for and identifying antibody) . (National Blood Transfusion Center) (Regional Blood Transfusion Center) ( ) .

(hepatitis B virus) (HIV) . ( ) (Specificity) (avidity) () (rouleaux forming properties) . . . (non immune source) (anti-A ) (artificially immunized) () . Dolichos biflorus A1 (anti-A1) Helix pomati helix aspersa A ( anti-A ) mouse hybridomas ( ) .

ABO (anti-A anti-B anti-AB) (affinity) ( ) (thermal) .

() (Immunization and plasmapheresis) :-

ABO .

(Blood group-specific substance) ( ## A B AB) . () (anaphylactic reaction) .

:-

- ( B C) (HIV1 HIV2) 6 6 . () 2 3 . ( ) .

- () (cold agglutinins) dialysis sodium azide ( anti-A anti-B anti-D ) . (500 ) (5 10 ) 2 8 .

- (- 20 ) 2 8 .

- (post-menopausal women) ABO .

1- :-

ABO MN () leguminosa Dolichs biflorus A1 (anti-A1) N-acetyl-D-galactosamine Helix pomati A (anti-A) Ptilota plumosa Griffonia simplicifolia fomes fomentairus B D-galactose O Ulex Europaeus H (anti-H) Angeilla anguille L-fucose M (anti-M lectin) Lberis amara N (anti-N lectin) Vicia graminae D-galactosyl .

- D (Preparation of anti-D reagent) :

anti-D D anti-D . anti-D ( ) ( ) . anti-D .

anti-D () . anti-D .

anti-D D (monoclonal anti-D) anti-D anti-D .

anti-D ABO . anti-D ( ABO) . 6 6 .

(genotype) D D dce/dce Dce/Dce Dce/dce ABO () .

(preparation of anti-human globulin) :- . (AHG) (Reference Laboratory) ( ) .

(Blood Substitute) . ( 2-6 ) . 42 . () . . ( ) :

1- .

2- .

3- .

4- (isotonic) .

5- .

6- () .

7- .

8- .

9- .

10- . (Possible Red Blood Cell Substitutes) :- . :

1- :-

(Bovine Hb) (Encapsulated Hemoglobin) (Modified Hemoglobin Solution) (Human Recombinant Hemoglobin) (Hemoglobin from transgenic animals) .

2- (Perfluorochemical emulsions) :

(perfluorocarbpnate) (Teflon) .

(Blood Substitute) . ( 2-6 ) . 42 . () . . ( ) .

:-

11- .

12- .

13- .

14- (isotonic) .

15- .

16- () .

17- .

18- .

19- .

20- . (Possible Red Blood Cell Substitutes):- . :-

3- (hemoglobin Solution):-

(Bovine Hb) (Encapsulated Hemoglobin) (Modified Hemoglobin Solution) (Human Recombinant Hemoglobin) (Hemoglobin from transgenic animals).

4- (Perfluorochemical emulsions):- (perfluorocarbpnate) (Teflon) .

ABO Blood Group System . ( ) . (1900) (Karl Landsteiner 1900) " " (ABO blood group) : (antigen) (antibody) "" (A) "" (B) "" "" (A or B antigens) . () (groups) "" (A antigen) "" (blood group A) "" (B antigen) "" (blood group B) "" "" "" (blood group O) " " (Decastello and Sturli) "" (AB blood group) "" "" (A and B antigens) "" (AB blood group) . "" "" (A & B antibodies) "" "" "" "" 3 6 .

"" "" "" "" N-acetyl-D-galactosamine "" "" D-galactose "" "" "" L-fucose " " H ABH blood group ABO blood group ABH . () .

ABH (Chemical nature of ABH antigens) :

" " glycosyltransferase. (catalyses) () . glycosyltransferase (fucosyltransferase) ( ) - (histo-blood group antigens) .

H (-fucosyltransferase) () - (L-fucose) 1-2 (1-2 linkage) - (D-galactose) H . H (H-transferase) 1 (type 1 chain) 2 (type 2 chain) 1 . H (Bombay phenotype) H - (para-Bombay phenotype) H . A (A-transferase) N-acetyl-D-galactosamine 1-3 (1-3 linkage) - (D-galactose) H B (B-transferase) 1-3 (1-3) - (D-galactose) H . A B 1 2 (substrate) A B H () H (H-transferase) () A B A B H ( H A B) .

L-fucose () (Immunodominant sugar) H O N-acetyl-D-galactose A D-galactose B 1 .

" " (epithelial cells) - ABH .

Gal1-R (type 1 or type 2)*

Gal1-R (type 1 or type 2)*

Fuc

A-antigenGalNAc1-3Gal1-R (type 1 or type 2)*

Fuc

Or

B-antigenGal1-3Gal1-R (type 1 or type 2)*

Fuc

Figure-1 Relationship of type 1 and type 2 chain oligosaccharides, the H-antigen, A-antigen and B-antigen of human histo-blood groups.

*Type 1 chain: -

Gal1-3GlcNAc1-4[Gal1-4GlcNAc1-3]n Gal1-R.

*Type 2 chain: -

Gal1-4GlcNAc1-4[Gal1-4GlcNAc1-3]n Gal1-R.

R= An asparagine-linked, serine/threonine-linked or lipid-linked (ceramide) glycoconjugated backbone.

n= 0- 5 or more

" " (ABH determinants) 6 (disaccharide chains) 1 6 2 .

Figure-2

Type 1: Gal1-3GlcNAc1-R

Type 2: Gal1-4GlcNAc1-R

Type 3: Gal1-3GalNAc1-R

Type 4: Gal1-4GalNAc1-R

Type 5: Gal1-3Glc1-R

Type 6: Gal1-4Glc1-R

- . .

:-

1. () (In vivo) .

2. (frequency) .

ABH (Development of ABH antigens) :-

ABH ( 50%) ( ) . A,B .

A A B 700000 B A,B 600000 A 700000 B AB B AB A A .

Bombay phenotype :-

A B ( O ) A B H . 1952 ( ) . H A B H A B (A and B transferase) . H h glycosyltransferase (L-fucose) . A B (very specific) H A B . A B . A B .

H (H antigen) H (anti-H) (routine ABO typing) O . ( ) H O . IgM .

H Ulex europaeus H (anti-H) A B AB .

Inheritance of Bombay phenotype (Oh or ABH null) :

H ABO ( ) h H H-glycosyltransferase H A B O ABO A B O O H H Oh ABHnull .

- ABO .

() A (Subgroups of A) :

ABO A 1911 Von Dungern Hirschfeld A A (anti-A) A . A1 (80%) A2 ( 20%) . A :

1. A (anti-A) A1 (anti-A1) A1 (anti-A1 lectin) Dolichos biflours .

2. AB (anti-A,B) .

3. Ulex europaeu H (anti-H lectin) .

4. A1 (anti-A1) .

5. H A .

AB A1B A2B .

A1 A2 A1 A ( A) A2 A N-acetylgalactosaminyltransferase A2 A1 A2 H A1 A1 A2 A A .

A2 A O A A,B ABO A O O A,B A O A,B (anti-A,B) A A O . A Aint A3 Ax Am Ael 3 . A / -A,B

(mixed field) -A,B -A (+1 - +3)

A3 Ax

A H H

Am Ael-3: A A / A,B B (Subgroups of B) :

() B A B A2 . B B3 Bx Bm B (anti-B) A,B (anti-A,B) .

- ABO 87 105 Immunohematology principle and practice by Eva D. Quinly- ABH (ABH Blood Group System In Disease) 95 .

- ( ) 95

- 97

The ABO Blood Group

The ABO blood group system was the first human blood group system to be discovered and remains the single most important system in routine transfusion and transplantation practice. The system was first described by Karl Landsteiner in 1900 (reported in 1901). Landsteiner drew blood from coworkers in his laboratory, separated cells and plasma, and mixed the cells and plasma from the various people on glass tiles. Landsteiner was able to identify three different patterns of reactivity, which he termed "A," "B," and "C." These types were later reclassified as group A, group B, and group O. On further testing, one of Landsteiner's associates discovered group AB (the rarest of the common ABO types) and reported this blood type in 1902. These four groups, A, B, O, and AB, represent the four major groups within the ABO system.

Why was the ABO system the first to be discovered, and why does it remain so important today? The answers to these questions can be explained by two simple principles. First, almost all normal, healthy people older than 3 to 6 months of age have naturally occurring antibodies to the ABO antigens that they lack. This has been termed Landsteiner's Rule. These antibodies were first called naturally occurring because they were thought to arise without antigenic stimulation. It is now known that this is not the case. Nevertheless, these naturally occurring antibodies are almost always present. Secondly, these naturally occurring antibodies are mostly of the immunoglobulin M (IgM) class. That is, they are antibodies capable of agglutinating saline-suspended or low-protein-suspended RBCs without enhancement and may readily activate the complement cascade. This assures that a potentially life-threatening situation may exist during the first attempted transfusion of a recipient if appropriate precautions are not taken to provide the correct ABO blood group for transfusion.

In its simplest form, the ABO system can be demonstrated by simple room temperature mixing of RBCs and plasma or serum. The reagents that are used in the laboratory for routine ABO typing may simply consist of serum from group B people (containing anti-A), serum from group A people (containing anti-B), and cells from known group A and B people. Alternatively, reagents produced by monoclonal technology may be used for ABO typing.

INHERITANCE

It was originally believed that the ABO antigens were inherited directly along with the ability to produce the antibodies to the ABO antigens not produced. This theory of inheritance of ability to produce specific antibody without antigenic stimulation was soon discarded after it was shown that the naturally occurring antibodies were actually caused by stimulation with antigenically similar substances present in our environment. This theory was proven in experiments with chickens that develop similar, naturally occurring isoagglutinins, antibodies that agglutinate blood cells of some people of the same species. When chickens were grown in an experimental "germ-free" environment, they failed to develop these naturally occurring isoagglutinins. Further work showed that the isoagglutinins of humans were developed in a similar way. This is why it generally takes 3 to 6 months for newborns to develop sufficient levels of these isoagglutinins to be readily classified. The term naturally occurring is most appropriately replaced throughout the remainder of this chapter with the term non-red cell stimulated (NRCS) when referring to these isoagglutinins.

The ABO blood group genes code not for the antigens directly, which are carbohydrate in nature, but for the production of glycosyltransferases. Glycosyltransferases are enzymes that facilitate the transfer of carbohydrate (sugar) molecules onto carbohydrate precursor molecules. The transferase associated with each blood group is specific for a particular immunodominant sugar. The immunodominant sugar molecule completes the antigenic determinant when combined with the precursor substance. A listing of the specific transferase along with the immunodominant sugar for each common ABO antigen is given in Figure-1.

The ABO genes seem to follow simple Mendelian genetic laws and are inherited in a codominant fashion because both A and B alleles are expressed when present. The O gene is an amorph or silent gene in that it appears to have no gene product. No specific transferase is associated with the O gene. The ABO locus is found on the long arm of chromosome 9. The A, B, and O genes may be found alternatively at these loci, one on each of the pair of chromosomes in any combination. Variants of A and B rarely may be found but are simple replacements for the more common gene and are inherited similarly to the more common forms of the A and B genes.

THE STRUCTURE OF THE HUMAN ERYTHROCYTE A1 AND A2PHENOTYPES AND THEIR DIFFERENCES

The human erythrocyte phenotypes Al and A2 are coded by distinct allelomorphic genes and determined by the presence of terminal a-1-3-N-acetyl-D-galactosamine (GalNAc) on oligosaccharide side chains attached to protein and lipids (e.g. Band 3 and ceramide) (31).

In 1911 Von Dungern and Hirschfeld (32) showed that not all blood group A erythrocytes are identical in terms of their A antigen. This observation led to the division of group A blood into the subgroups now known as Al and A2. These are characterised by their agglutination patterns shown with anti-A and anti-Al antibodies and anti-Al lectin from Dolichos biflorus (33,34). Accordingly group AB blood can be similarly divided into subgroups A1B and A2B. Anti-A from group B subjects is said to contain two components, anti-A and anti-Al. Also, Al erythrocytes are said to carry A and Al antigens, while A2 erythrocytes carry A antigen without Al (33,35). However, Issitt (8) considers that the failure of anti-Al from group B serum to agglutinate A2 erythrocytes relates not to its specificity, but to the restricted number of copies of the antigen that define A2 erythrocytes. Nevertheless, division of A erythrocytes into the major subgroups Al and A2 is universally accepted. However, the controversy is not finished about the nature of, and the immunochemical basis for, the difference between A1 and A2 erythrocytes Other blood group system 200 . 19 (High frequency antigens) (Low frequency antigens) (Private antigens) . () :

1. .

2. .

( 118 )

Lewis blood group system:-

1946 Mourant anti-lea (Mrs. Lewis) . anti-leb 1948 . Lea (LE1) Leb (LE2) Lec Led Lex (LE3).

:-

1- .

2- Lele Hh Sese .

3- ABO.

( ) Lewis system phenotype and antigens ABO (glycoprotein) . Lea Leb . () :-

1- Le ( a + b - ).

2- Le ( a - b + ).

3- Le ( a - b - ).

Le ( a + b + ) .

Lele 19.

Lewis antigens development . Le (a - b -) Lea . Lea . Leb . Le (a - b +) Le (a + b +) ( ). Le (a - b -) Le (a + b -) Le (a + b+) Le (a - b -) . :-

Le (a- b-). .

Lewis system antibodies

anti-Lea anti-Leb 1. ANTI-Lea:-

anti-Lea ( ). Le (a - b +) Lea Leb anti-Lea. (anti-Lea) Le (a - b -).

anti-Lea IgM IgG. 37 (. anti-Lea (invito hemolysis). (enzyme-treated-RBC) ( ) .

2. ANTI-Leb:-

anti-Leb anti-Lea IgM () . anti-Leb Le(a- b-) Le(a+ b-) . .

anti-Leb (soluble Lewis substance).

anti-Leb anti-LebH anti-LebL. anti-LebH Le(a+) O A2 anti-LebL Le(a+) ABO.

anti-Lec anti-Led () Le . Clinical Significance of Lewis antibodies:

1- Hemolytic Disease of New Born (HDN):-

IgM . IgG .

2- Hemolytic Transfusion Reaction ( HTR):-

(antglobulin phase) () . The kell, Duffy and Kidd blood group system:-

:-

1- ( ) :-

2- IgG.

3- (Worm antibodies) 37 .

4- (HDN) (HTR).

5- (AHG). .

Kell blood group system :-

Kell (anti-globulin test) (Coombs test).

Kell Rh . k Kpb jsb KEL11. IgG :- Anti-K, anti-k, anti-Kpa, anti-Kpb, anti-Jsa, anti-Jsb, anti-WKa, and anti-WKb .

anti-K HDN HTR K D ( ).

. K (anti-K) (HTR). . 47 10 K. . .

Duffy blood group system:-

Fya Fyb Fyx Fy 1. Fya Fyb (phenotype):-

1- Fy (a + b +).

2- Fy (a + b -).

3- Fy (a - b +).

4- Fy (a - b -).

.

Fy (a - b-) Fya Fyb (P. vivax and P. knowlesi) P. falciparum FY. Fya Fyb P. vivax .

Duffy system antibodies:-

anti-Fya anti-Fyb (AHG) IgG . anti-Fy3 anti-Fy4 anti-Fy5 Fy3 Fy4 Fy5 .

:-

. .

Kidd blood group system:-

Jka Jkb. :-

1- Jk (a + b -).

2- Jk (a + b +).

3- Jk (a - b +).

4- Jk (a - b -).

. .

Kidd system antibodies:-

. IgG IgM anti-Jka anti-Jkb anti-Jk3.

:-

() AHG.

I blood group system :-

I ABO . i I. I I i I. I I i i I .

I I blood group system antibodies :-

anti-I anti-i IgM (naturally occurring antibodies) ( anti-I) 4 .

I (autoantibodies) (cold autoimmune hemolytic anaemia) (cold agglutinin disease).

I:-

I (HDN) IgM . anti-I anti-i (Hemolytic anaemia) (destroy transfused cells) (autologous cells).

P P blood group system:-

P P1 P2 A1 A2 ABO. P1 P P1 P2 P P P1 . Pk.

P ( ) :-

P1 P2 P1k P2k P.

P ABO D-Galactose N-acetyl-Dgalactosamine N-acetyl-D-glucosamine .. P1 P2 ABO () (glycosyltransferases).

P blood group antibodies:-

P P1 (anti-P1) P2 P P1k P2k P IgM . P (autoanti-P) paroxysmal cold hemoglobinuria (PCH). Ecchinococus.

P:-

P .

MNSs :

MNSs MN Ss 4. M N D-galactose N-acetylgalactosamine Sialic acid (Sialic acid) M N.

M N S s U. M N M N MN. :- MM NN NM. S s ( MN Ss ) :-

Ms MNS MNSs MNs NS NSs MS MSs MN . M N ABO . M N (homozygous) MM NN M N (heterozygous) .MN S s . MN .

S s M N M N S s . S s U . U ( Rh null) S s. MNSsU Mk .

MNSsU :-

M (anti-M) N (anti-N) IgG . S (anti-S) s (anti-s) IgG U (anti-U) IgG . anti-M anti-N anti-S anti-s.

Xg Xg blood group system :-

Xga Xg(a +) Xg ( a -) X chromosome X (Hemizygous) (Homzygous) (Heterozygous). Xga Xg ( a + ) X . Xga ( ) Xg ( a +).

Xga 88.7% 65.6% XX XY.

Xg :-

Xg ( ) X Glucose-6-Phosphate dehydrogenase.

Lutheran blood group system:-

Lua Lub :-

1- Lu (a + b +).

2- Lu (a + b -).

3- Lu (a - b +).

4- Lu (a - b -).

Lua 94% Lub 6% .

:-

:- Lua ( anti-Lua ) Lub (anti-Lub) IgM . anti-Lub anti-Lua (AHG).

:

1- .

2-

3-Immunohematology principle and practiceRh BLOOD GROUP SYSTEM ABO (ABO) (Pretransfusion reaction) . .

(Landsteiner) (Alexander Wiener) 1939 (Rhesus monkey) (Rabbit) (guinea pig) ( ) 83% 17% . 83% (anti-Rh) . (Rh positive) (Rh negative) . 1939 (Levine and Stetson) (stillborn baby) ABO (fatal transfusion reaction) (irregular antibody) 83% . (New blood group system) () Rhesus (Rhesus system) . (Specificity) anti-LW Rh (anti-Rh) Rh-D positive () anti-D Rh-D negative D .

Rh ABO A B Rh 5 (6 d D) C, c D, d, E, e Rh ABO (gene enzyme action) Rh .

* Rh 83 85% 40 6 . D Rho ABO D (Strongly antigenic) ( ) 50% (Rh ve) D (anti-D) Rh +ve -D (anti-D) (pretransfusion reaction) .

Rh +ve Rh-D ( ) Rho ( ) Rho (D) ( ) Rh -ve D Rh-D Rho Rho(D) -D (anti-D) D . CDE cde/cde . 3 3 . Rh 8 Halotypes . 3 Cc Dd ee C c D E e . d . D . Rh : Rho rh' rh" hr' hr" rh (' ) Cc Ee rh' C rh" E Rho D r D . (Rh) : Rh1 D Rh2 C (1) .

Rh ( 135 149) . (1) RhROSENFIELDWIENERFISHER-RACE

Rh1

Rh2

Rh3

Rh4

Rh5

Rho

hr"

hr'

rh"

rh'D

C

E

c

e

Du D Weakened Expression of the D antigen Du )) :-

Du D Rh(D) -ve +ve Du (Bovine albumin) - D (modified anti-D antisera) Rh (D) -ve. ( Du) :-

1- D (incomplete D antigen).

2- () (position effect).

3- D (inherited gene coding for weakened D antigen expression).

(Du antigen) Cde/cDe Cde/CDe.

Du :-

1- Du ( Du) D Rh(D) -ve +ve Du Rh(D) -ve Du D D (D mosaic) D . D (anti-D) Du .

2- Du Du Rh(D) -ve Rh(D) +ve D Rh D ve Rh Du +ve .

D ( antigen Du) D D D . (Rh -ve) . D (anti-D) position Du .

* Rhnull ( Rhnull) :-

Rhnull Rh ( Rh C, c, D E, e) - - - / - - - Hro ( Rh:17) Rh:29 Rh:29 Rhnull . Rhnull X1 DCE Rhnull :-

1- Rhnull Regulator- X1 .

2- Rhnull Amorph - DCE.

Rhnull Rh-29 (anti-Rh:29) Rh . Rhnull. ( Rhnull) Rhnull . Rhnull Rhnull .

Rh Rh Rhnull (Hemolytic anemia) . . Rhnull (Hb and Htc) () (Stomatocytic spherocytic) .

The LW Antigen LW Rh (Rhesus monkey) Rho(D) antigen LW Rho(D) . LW . LW .

LW D +v D -ve. LW Rhnull .

Rh system antibodies :-

Rh +ve Rh -ve ABO . ABO ( ABO IgM) Rh ( stimulation) ( ) G (IgG) ( IgG1 IgG3) 6 6 . 0.25 0.5 . Rh Rh Rh . (extravascular) .

Rh (Rh Immune Globulin = Rh Ig) :-

(Hemolytic Disease of New Born = HDN) (Rh -ve) (Rh +ve) (stillbirths) (spontaneous abortion) . () ( 6 6 ) . (Rh +ve) ( ) (anti-D) G (IgG) IgG1 IgG3 (IgGs ) (Rh-D antigens) (Rh +ve) .

Rh Ig HDN . Rh Ig G (IgG) -D (Human IgG anti-D) ( ) (antipartum dose) 72 .

Rh Ig (Rh Ig coat the D antigen sites of fetal RBC) . Rh Ig (antipartum) 72 (postpartum) .

[Rho (D)] Du (antigen Du) D . Rh Ig D C c E e HDN (Rh antibodies) IgG HDN .

/

2001

Rh-D Grouping (Saline Technique)

Rh ABO (Rh-D negative) (Rh-D positive) (Rh-D +ve) (Rh-D -ve) (anti-D antibodies) (Hemolytic anaemia) . (Rh-D group) . .1- Slide Testing : 37 . 40 50% . D (anti-D) ( ) .

2- D (Microplate testing) :

.

- 96 () (96-well microtiter plate) .

3- Tube Testing:- Principle:- (Rh-D) (antiserum incomplete) D 3 5% (saline). 37 .

D ( anti-D ) saline anti-D albumin anti-D D ( complete antibodies ) () (saline) incomplete antibodies)) (sensitize) bovine albumin (proteolytic enzyme)

Material and reagents:-1- Incomplete anti-sera (anti-D serum)

2- ( 5% ( saline )

3- (control) (Rh-D +ve) (Rh D -ve).

5- 10 75 .

6- .

7- 37C .

8- .

Method :-1- (100 ) D (anti-D serea ) 10 75 5% .

2- 37 C 15 .

3- 1 3 ( control ) .4- 15 3600 rpm sero-centrifuge . -Du ( serum anti-Du ) - Du .

D (Antigen Du Testing) :- . - -Du indirect antiglobulin (Coombs) test -Du (anti-Du serum) Du- -D (anti-D serum)

Material and reagents:-

1- Anti-human globulin (AHG)

2- Anti-Du.

3- Rh ( D) group Method:-

1- (100 ) 5% (200 ) -Du (anti-Du serum) .

2- (control) .

3- 37C ..4- (saline) .

5- AHG . 5 ..6- .

/

2001

(Lectin) :-

( ) (in suspension) ( ) .

(Legumes and Ricinus communis) ( ) Colonisatin Factor antigen E. coli.

1888 Hermann Stillmark Tarut . ABO MN () leguminosa Dolichs biflorus A1 (anti-A1) N-aceytyl-D-galactosamine Helix pomati A (anti-A) Ptilota Griffonia simplicifolia fomes fomentairus B B D-galactose O Ulex Europaeus H (anti-H) Angeilla anguille L-fucose M (anti-M lectin) Lberis amara N (anti-N lectin) Vicia graminae D-galactosyl .

Properties of lectin :

. 1- (monosaccharides) ( di- and trisaccharides ) .

2- .

3- .

.

(Preparation of lectin extract) :

1. 2 25 PBS 7.4 (pH 7.4) 20 24 4 .

2. .

3. (supernatant) 6000 30 .

4. (supernatant) (0.7 mm) .

5. ABO :-

1. ( ) 1:1 1: 1024 100 100 PBS .

2. 100 3 5% PBS ( 3 PBS pH 7.4) 60 .

3. .

compatibility tests ( : Pretransfusion Testing)

() () (ABO) (Rh D or Rh factor) . . ABO (Rh D) ( ) .

(DONOR TESTING):-

: ABO 1 2 (HIV1 & 2) (syphilis) (HBsAg & HBcAg) (HCV) (cytomegalovirus) (selected antigen typing) . (Choice of Donor Blood):-

ABO RH . (Packed RBCs).

Rh () ( D) D. D .

O neg . . .

. .

ABO (Packed RBCs)

OOO___

AAAO__

BBBO__

ABABABABO

.

Crossmatch Testing:-

: () (Major crossmatch) . () (Minor crossmatch) . () . :-

1. .Immediate spin in saline at room temperature.

2. 37 0 .Incubation at 37C with enhancement medium.

3. () -1.Antiglobulin phase after washing incubated cells with caline.

Compatibility Test Cross-match Test (Cross-match) (Compatibility) :-

1. (HBV) (HCV) (HIV) (Syphilis).2. ( ).

3. ABO (Rh factor) .

4. (clinically significant antibodies) .

5. (crossmatch) .

(pretransfusion tests) :

1. .

2. .

3. (sensitivity) (specificity) (simplicity) .

4. .

- .

:-

. .

:-

. anti-Lea anti-Leb (autocontrol) ( ).

3 . . . . ( ) . 10 .

:-

.

:-

(control) .

Cell suspensions:-

1.5% NISS 3 5 % . ( ):-

:- 30% Bovine albumin ( ) Anti- human Globulin.:-

1- ( ) 8.5 / (0.85 % saline). 2- ( ) .3- 30% Bovine albumin.

4- Antiglobulin serum.5- (control).

6- 10 x 75 .

7- 37C.8- .

Principle:- 30 % bovine albumin antiglobulin serum . Method:-

- Bovine albumin:-

1- .2- 30 % bovine albumin 37C 45 . 30 1000 rpm .- antiglobulin serum :-1- 5% . 37C 45 .2- 1000rpm . ( ) :-

3- Saline .4- (anti-human globulin serum) 5 . 1000 rpm . .5- (control) saline 1 4.6- ( ) 1000 rpm .:- 0.1 0.2 .

Blood Bank Practical (spring 2002)

Dr. A. M. Milad

Preparation of anti-A1 Sera

Anti-A1 reagent is necessary for the routine distinction of A1 cells from A2, but it is very expensive to purchase commercially. It can be produced by the absorption of anti-A from the serum of a group B donor (which has anti-A + anti-A1), using A2 cells. This leaves anti-A1 which is referred to as "absorbed anti-A (anti-A)".

Anti-A1 lectin may also be produced from the seeds of Dolichos biflorus, a plant grown in tropical and subtropical countries; both seeds and plants can be purchased inexpensively. The reagent prepared from the seeds is potent and stable. After the soaked seeds have been ground in a mortar or liquidizer, the reagent is extracted using sodium chloride, with centrifugation and prolonged cold precipitation (4 C for 3 months). Alternatively, 0.31 mol/litre (23 g/litre) glycine and 0.31 mol/litre (18 g/litre) sodium chloride may be used. In this case, the filtered extract is further diluted with the same solution of glycine, if necessary. This is followed by heating at 56C for 30 min, freezing overnight, thawing, and cold precipitation at 4C for 3 weeks.

The reactivity with A2 cells, which occurs in higher dilutions of the extract obtained by either of these methods, can be overcome by further dilution of the extract or by absorption using A2 cells.

Preparation of anti-A1 Sera from the Serum of Group B Donor:

Principle: -

When A2 cells incubated with group B serum. A2 cells will absorb anti-A from the B serum and leaves anti-A1. The absorbed serum will agglutinate A1 but not A2 cells.

Material and Reagent: -

1. Blood group B serum (preferable with anti-A titer).

2. Washed, packed, A2 cells and 3-5% washed, suspension of A2 cells.

3. 3-5% washed suspension of A1 cells.

4. Four 10 x 75 mm tubes and pipettes.

Method: -

1. Mix two volumes of B serum with one volume of washed, packed, A2 cells in a 10 x 75 test tube, and incubated at 4C for 1h.

2. After the incubation period, centrifuge the mixture at 1500 x g for 10 min and collect the supernatant serum.

3. Check the collected serum for its agglutination ability against A1 and A2 cells. If the serum gave any reaction with A2 cells, reabsorb the serum with half volume of washed, packed, A2 cells.

Write a full report about your result.

:

Investigation of the ABO Blood Group System

Landsteiner discovered the ABO blood group system by mixing together blood cells and sera from his laboratory staff.

In this laboratory session you will repeat Landsteiner's experiment. It is not possible to reproduce the state of knowledge which existed before Landsteiner's work since we are well aware of the characteristics of the ABO blood group system and can never look at the results of similar experiments with an open mind.

However, even with this knowledge you will see that ABO grouping without reagents is not as straightforward as it would seem. During the discussion of the results, which you obtain, you will see that there are several circumstances, which might have led to different conclusions from Landsteiner's work.

Practical Work

You are supplied with a number of labelled samples of red blood cells, with their corresponding sera similarly labelled. Carry out a 'chessboard' cross comparison between each sample of serum and cells. Allow the tubes to remain at R.T. for 60 min. before reading the results and deciding to which ABO group each sample belongs.

In your full report of this investigation, explain how you decided to which group each sample belonged.

() transfusion reaction () . (double-check system) . . :-

1- ( ) .

2- (control) .

3- ( ) .4- .

5- O A B ( dangerous O blood group) .

6- .

7- .

8- (quality control) .

9- , .* :-1. .

2. .

3. .

4. .

5. (drugs/ anti-drugs complex) .

6. (pyrogens) .

7. foreign debris trace metals .

:-

1- Hemolytic transfusion reactions :-

1667 J Denis Denis Denis 150 .

(intravascular) (extravascular) :-

- ()

2- () .

:-

- (Immediate intravascular hemolysis) :-

(intravascular) (complement) IgM . ABO ( ) A2 A2B . O . A B (anti-A or anti-B) ( ) .

(Sings and Symptoms) :-

( 40 ) (shock) (haemoglobinuria) (oliguria) (anuria) . (pain along the infusion site) . :-

(1) (Disseminated Intravascular Coagulation) (thromboplastin) .

( 2) 3 :-

- .

- 20 50 .

- .

- (Delayed extravascular hemolysis) :-

(extravascular) IgG ( anti-D MNSs) (Macrophages) ( ) 7 14 .

:- (dark urine) (Bilirubin) . .

- (Pseudohemolytic Reactions) :-

(haemoglobinuria) (elevated plasma hemoglobin) . . .

:-

1- .

2- () (water used for irrigation in the circulation) .

3- ( 50 ) .

4- (glycerolization) (deglycerolization) .

3- .

6- .

7- , .

- (Patient conditions that may cause hemolysis) :-

. G6PD Clostridium infection Paroxysmal Nocturnal Hemoglobinuria sickle cell disease autoimmune hemolytic anaemia . .

2- () (Plasma Component Reaction) :-

(allergic reaction) .

(Allergic Reactions) :-

IgE ( allergen) IgE (IgE fix to mast cells and basophils) (Histamine) (vasoactive amine) (hives) (hay fever) (sever itching) . anti-histamine . . . (anaphylactic) () IgA ( IgA) .

- .3- ( ) (WBCs Antibodies Reactions and Pyrogenic Reactions):- 257 258.

4- (Bacterial Reactions):- 259 260.

5- (Cardiovascular Reactions):- 261.

6- (Embolic Reactions) :- 272.

7- (Citrate Toxicity) :- 262.

:- 263 . (Investigation of Transfusion Reactions) :-

() ( ) .

:-

. .

:-

:- .

1- (Serological tests) :-

:-

- (pre-transfusion samples) ( ) .

- (clotted samples) (post-transfusion sample) 24 .

- .

- 24 (methaemoglobin) .

:-

(Rh) ( pre- and post-transfusion samples) - (direct antiglobulin tests) ( ) - ( ) - . A B O A B (anti-A & anti-B) 128 O (dangerous group O donors) . O .

2- (Bacteriology tests) :-

(culture) 4 20 32 Gram staining and smear test examination .

3- (Biochemical tests) :-

(bilirubin test) (free hemoglobin) (hemoglobin derivatives) . .: : * 3 99.9% :-1- A2- B3- AB O

A B A B B Rh Rh . + A A+ . 0.3 2 % . . 17 .. (..etc D,C,E,c,e,M,N,S,s,P,le,K,k) Rh .. . / . . . 3 10 . ,. . . () . 3 :1-2- D Rh <