BLOOD BANKING study of immunologic properties and reaction applied in blood groups and antibodies Antigen a foreign substance

ce which when introduce into body stimulate antibody production Antibody a protein produced by plasma cells a specific substance present in response to an antigen collection, preparation, preservation, processing and distribution of blood components and derivatives comprises blood banking 450-500 mL blood + 63 mL anticoagulants 1. 2. 3. 4. 5. 6. 7. 8. 9. BLOOD COMPONENTS Packed RBC Fresh frozen plasma Plasma Cryoprecipitate Cryosupernate Platelet concentrate Platelet rich plasma Leukocyte concentrate/granulocyte concentrate Leuko-poor RBC

Mechanisms of blood typing, compatibility test, Coombs test/ Anti-Human Globulin Test, screening of serums of a typical Ab, bleeding of donors for blood transfusion, bleeding technique, proper disposal of blood based on quality assurance IMMUNOHEMATOLOGY Immunologic reaction applied in blood bank and transfusion service GENETICS Study of inheritance; transmission of inherited character from parents in offspring IMPORTANCE OF GENETICS IN BLOOD BANKING Parentage testing When seeking an explanation of unexpected reaction observed in the laboratory and when asking an additional test in order to resolve the problem

Transfusion reaction hemolytic transfusion reaction (example), circulatory overload, anaphylactic urticaria (itching hives) Parentage testing test genetic markers Types: 1. Direct Exclusion Genetic marker/gene in child/absent in parents e.g.

O Oh

O O

child B B Obligatory gene

Exclusion Non-exclusion

Bombay phenotype

2. Indirect exclusion Mother involved; mother transmits gene its child but gene absent in father e.g.

+ +

JK (a b )

JK (a b ) Genetic marker

Child JK (a+ b-)

Non-exclusion

Key blood group system 2 KINDS OF CELL DIVISION 1. Mitosis same number of chromosome (46 in man) XX- XY - Chromosome Made up of DNA which carry genes and protein Nucleotides a. b. c. Units of DNA Deoxy Purine Ah Pyrimidine, CT

2. Meiosis half of the number of chromosomes of parents Crossing over Exchange of genetic information; closely linked genes on chromosome rarely cross over e.g. B group, MNSS

AMF AMm

AFF AFm

AMF AMm

AFf AFm

AMF

AMm

AFF

AFm

AMF

AMn

AFF

AFm

AMF

AMm

AFF

AFm

AMF

AFm

AMF

AMm AMn

AMm

AMF

Proteins Product of gene action DNA (template)

mRNA genetic message transcribed (3-letter cocon) cytoplasm (protein synthesis)

tRNA (carries Amino acids to mRNA surface) = polypeptide bonds TYPES/MODES OF INHERITANCE Autosomal Occurs in equal frequency of male and female not inherited on sex chromosome Codominant 2 genes (allelic/allele/all amorph = an alternative form of a gene which occupies a locus) in loci differ but are both expressed e.g. genotype AB Dominant e.g. histocompatibility Ag; ABO 1. Autosomal Dominant/Codominant Directly from parents; characteristics never ships as is always transmitted e.g. AB
AB B B

phenotype AB

AB

2. Autosomal Recessive e.g. serodermapigmentation, cystic fibrosis heterozygous parents (apparently not affected) e.g.

 must be homozygous in the allele to have the trait expressed parents may/may not expressed the trait e.g. XX Xx

XX Rare gene

Xx

Xx

Xx

if the recessive occurs in only 1 generation, not occurring in preceding or successive generation if the child (homozygous for recessive rare gene, they are blood relatives) e.g. hh = Bombay phenotype 3. Sex-linked Dominant Inherited from chromosome Genetic markers not transmitted from father to son, but must be transformed from father to his daughter to her son Color blindness, hydrocephalus, hemophilia A, B XY Affected male XY XY XX XX normal female XX XX

XX

XY

XY All sons normal all daughters affected

XY

xx

XX

4. Sex-linked recessive Queen Victoria 1st case with hemophilia gene (transmitted gene to Persia, Russia, and Spain) Affected male normal female carriers

 BLOOD GROUP SYSTEM ABO Blood Group System Major blood group in humans; made up of groups A, B, AB, O Names determined by the presence/absence of isoantigens on the surface of the RBC membrane and presence/absence of corresponding isoantibodies in the serum/plasma Discovered by Karl Landsteiner To ok blood samples from laboratory staff, made an RBC suspension and mixed it whith plasma or serum Discovered that only 2 Ag are responsible for blood type No agglutination = Ag present and Ab absent RBC Ag A (41%) B (10%) O (45%) AB (4%) A B None A, B Ab Anti- B Anti- A Either None Serum Plasma

Discovered in 1902 Landsteiner Law The antibodies present in serum or plasma when the corresponding antigen is not present in erythrocyte Mendelian Law A and B inherited antigens which arent fully developed until 3 years old; maximum reactivity is attained at late teens/early 20s. newborns exhibit low reactivity/titer of Ab once maximum reactivity is attained, it remains stable throughout A substance/B substance For bacteria and plants For stimulations of Ab production 1. Demonstrated as early as second month of fetal life 2. Ag age appears weak at birth at the age of 1 year, iso- agglutination reach full strength 3. ABO antigen persist through life altered 4. ABH antigen can be found in bacteria and other species 5. ABO antigen can be found in saliva, gastric and pancreatic secretion of secretors

 New born infants doesnt posses antibodies, antibody appears during post natal life Individual agglutination titer begins at late teens or early 20s exhibit or gradual increase and gradually decline Stable at 10 years

Characteristics of ABO groups 1. New born dont posses anti-A and anti-B if ever present they may have originated from mother through placental linkage 2. Developed in 3-6 months after birth 3. Reacts better at room temperature 4. They can be present in animals as (cels), in plants (lectin) 5. Chemically immunoglobulin 6. Since they are gamma globulin, they can expected to be very low titer, even absent in cases 7. Acquired congenital gammaglobinemia/hypoglobulinemia

Occurs in two forms: 1. Naturally occurring IgM not capable of crossing in the placenta (HDN) 2. Immune Ab Not normally present bit can be acquired Incompatible pregnancy and transfusion IgG capable of crossing the placenta

ABO subgroups Variation of the Ag that make it different to other subgroups but chemically similar to them Mistaken as group O To avoid misinterpretation and misidentification of blood group of a person If present, it is dangerous to the donor

Group A In 1911 Based on the reaction of Anti-A1 and Anti-A2

Anti-A A Red cells A Red cells + +

Anti-A1 + -

Blood group A1 A2

Ag A1, A A

Both qualitative and quantitative A1 is very potent gene that elicit high concentration of -3-N-acetylgalactosamyl transferase that creates 810-1,170,000 Ag sites of adult A1 red cell A2 is less difficient inheritance; 240,000-290,000 Ag sites of adult A2 red cell All A1 Ag are converted to subgroup A1,H-Ag is not present or hidden

A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 H H H

A H A H A H A H A H A H A H

A1 Weak A subgroups Ao, Ax, Am, Ay, Aenol, Ael, Aint

A2

Can be serologically differentiated by the following: 1. Forward grouping of A and H Ag using anti-A, anti-AB 2. Serum grouping of ABO isoagglutination and presence of anti-A1 3. Absorption elution test with anti-A 4. Saliva testing to detect presence of A,H angtigen

Group B Use anti-A and anti-AB to differentiate B1, B2, B3, Bm, Bx, Bw, Bel (Bandeiraea simplicifolia or modified seed extract that agglutinate human RBC with sime known specificity BS, - lectin) for differentiating group B variants; differentiate acquired B like Ag from true B Ag; agglutinate true B-Ag

Weak B: Criteria for differentiating 1. Strength of agglutination with anti-B, anti-H, anti-AB 2. Presence or absence of ABO isoagglutinins in the serum 3. Adsorption/elution using anti-B 4. Presence of B substance in saliva Subgroup AB= A1B & A2B Newborn A1 appears as A2B Reagents to determine ABO subgroups: Anti-A Reacts with cells and subgroups of A

Absorbed Anti-A (Anti-A1) Reacts with A1 only

Anti-Ab Reacts with Ao, Ax, Arm, aside from A, B, isoagglutinogen

Anti- A1 lectin Derived from Dolichos biflorus ; reacts with A1 and A1 B

Anti-H lectin Derived from Ulex europaeus; reacts with A2 & O

Detects ABO (weak) Confirm anti-A, anti-B

Anti-AB

Serum Grouping Serum A (Anti-B) B (Anti-A) AB (both) O (both) A cells + + B cells + + AB cells + + + O cells Blood groups A B AB O

Example: 1. (+) to O serum = A, B, AB (-) to A cells = A, AB (+) to anti-A = A, AB (+) to B cells = A, O 2. (+) to B serum = A, AB (-) A cells = A, AB (+) O serum = A, B, AB (-) AB cells = AB

IgM Reacts with room temperature, doesnt cross the placenta

IgG Most predominant in group O

ABO DISCREPANCY Red cell test dont agree and discrepancy are technical in nature and serum test 1. Weak and missing Ab 2. Due to weak and missing ABO Ag 3. Protein/plasma abnormalities 4. Miscellaneous abnormalities

1. Weak and missing Ab The newborn = do not possess Ab Elderly patient = decrease in titer Leukemia Lymphoma Immunosuppressant drugs 1. T activation occurs upon exposure to hidden erythro Ag (viral infection) (bacterial infection) cause of removal of sialic acid in cell membrane expose hidden erythro Ag

(NANA) N-acetyl-neurabimic acid Group of sugars attach on protein backbone the major negative change covering the receptor; found on red cell

T ag

2. Tn activation Permanent, sialic acid (insufficient adhesion during erythrocyte maturation) Associated in Acquired A Phenomenon from Proteus mirabilis and asepticemic infection Hematologic abnormalities (thrombocytopenia) 3. Cad polyagglutinability CAD erythrocyte are polyagglutinate because they carry SDA antigen

SDA antigen Cant destroyed by enzyme treatment

Acriflavin Yellow dye for the anti-B reagent

Cis-Ab Unequal crossing over

RH BLOOD GROUP SYSTEM Role in HDN, transfusion Group of red cells with greatest significance (aside from ABO) More complex than ABO More than 40 different Rh Ag From Rhesus monkey lead to Rh discovery

Levine and Stetson 1939 The first human example

1940 by immunizing rabbit/guinea pig with Ab

Weiners and Peters Found anti-Rh in Rh who received ABO transfusion Rh + blood Rho (D) or Dag MAIN FACTORS 1. Rho (D) Most important immunogenic, most clinically important Classify as Rh+ or Rh- regardless of other Rh antigen 2. rh (C) 3. rh (E) 4. hr (c) 5. hr (e) D>c>E>C>e Rh antigen lipoprotein, integral part of red cell membrane low molecular weight (174,000) located at chromosome 1 (Rh locus) along with the gene for elipto cytosis
6-phosphoglutamate

ABO Carbohydrates

O gene Acting as autosomal dominant

More than 1 systen of nomenclature to define Rh blood group system 1. Rosenfield/Alpha/numeric Aims to bridge the confusion with fisher-Race and Weiner

 Describe a system which the reaction with a particular anti-sera Rh:1 e. g. D+ C+ E+ ceRh: 1,2,3,-4,-5 Rh:51

Rh:1 Rh:2 Rh:3 Rh:4 Rh:5

2. Fisher Race/DCE Indicate inherited 3 closely linked genes from parents assumes there are 3 closely linked loci each with a primary set of allelic gene Paired chromosome FR D C E D c E amorph gene antithetical >2 ag controlled by a pair of allelic genes D C E c e Genes closely linked in the chromosome, passed in sets of 3 8 possible combination 1. Dce 2. DCe 3. DcE 4. DCE 5. dce 6. dCe 7. dcE 8. dCE D, c, e D, C, e D, c, E D, C, D c, e C, e C, E C, E Ro R1 R2 R3 r r r ry Rho Rh1 Rh2 Rh3 rh rh rh rhy Rho, hr,hr Rho, rh,hr Antigens Weiner Agglutinogen Blood factors

3. Welner/Rh-h Can convert to Fisher; a gene at a single locus on each chromosome control the entire Rh system Each gene usually produce 3 gene on agglutinogen Weiner Rho rh

FR D C E c e

Rosenfield 1 2 3 4 5

rh hr hr

Gene Ro

agglutinogen Rho

blood of Rho, hr,hr

EXERCISE: 1. Rh inheritance with FR. Nomenclature Rh genotype of mother Rh genotype of Father Possible Rh genotype of offspring 1. DCe/Dce 2. DCe/dCE DcE/dce DCE/dCe

DCe/Dce

DcE/dce

1. DCe/DcE 2. DCe/dce 3. DCe/DcE 4. Dce/dce

2. Weiner 1. R 2. R2Ro 3. /r

FR DCe/dce DcE/Dce dCE/dce

Rosenfield Rh: 1,2,-3,4,5 Rh: 1,-2,3,4,5 Rh: -1,2,3,4,5

Anti-C + +

Anti-c + + Cc Cc CC

3. Determine Rh blood type 1. (+) to anti- Rho (-) to anti- rh (-) to anti - hr 2. (-) to anti-D (-) to anti-C (-) to anti-E (-) to Du 3. (+) to anti-D (-) to anti-C (-) to anti-E (+) to Du (D) (c) (e) Rh+(Dce- Rho) du+ (D) (C) (E) (d) (c) (e) Rh-(dce-rh) duRh+(DcE-Rhz)

4. Determine the phenotype and genotype Phenotype Anti-D + + + + Anti-C + + Anti-E + + Anti-c + + + Anti-e + + + FR DCce DCCe DcEe DcE Weiner Rh rh Rh Rh Rh2rh Rh2 Rh2

Probable genotype FR DCe/dce DCe/DCe DcE/dce DcE Weiner R R R R2

FINALS D factors and other variants

Cu, Cw, Cx, Eu, Du

Cw Variant of C Du Variant of D Red cell sample carrying O Ag Weak form of D Ag detectable only by indirect Coombs test (Anti -human globulin test) Rarely found in Caucasian but more common in Blacks It is not different Ag Designated as D+W Du RC +anti-D negative/weak reaction

Mechanisms expressing weaked D Ag 1. C-trans The inheritance of C in the transposition to D result in the weakened expression of D Ag 2. Mosaic/Partial D 3. Genetic Du

Genotype: Dce/dCe

D c e

d C e C in transposition to D Du phenotype occurs

C in cis position D Du doesnt occur/ Normal D

D C e

d c e

2. Mosaic/Patial D Composed of 4 fragments If 1 or more portion of Ag is missing the remaining portion can be weakly expressed Alloantibody Upon expose to foreign antigen produced by a similar species 3. Genetic Du The Ag are all present, but weakly expressed 2 GRADE OF Du 1. Low Grade Du Detectable by indirect Coombs test 2. High Grade Du Agglutinated by certain Anti-D

Indirect Coombs Test Y Y Y RBC + Anti- D centrifuge (15 secs)

Incubate (15-60 minutes at 37C )

Agglutination Rh+

no agglutination Rh-

wash 3x with NSS after the last washing + 2 gtts. AHG Centrifuge (15 secs) Observe agglutination

Agglutination Du+

no agglutination Rh-

Preliminary screening (Rh-)Du+ = patient RhDonor Rh+ Rh deletion Red cells- C,E are absent (_D_/_D_) Rh null All Rh Ag are not expressed on the Rc (___/___) Rh moderate All Ag are present but weakly expressed on the RC Rh deleted RBC morphology Rh antigen Red cell Survival Normal Increase D Ag/RBC Normal Rh Null Stomatocytes, sperocytes Absence of D,C,E,c,e reduced

METHODS OF Rh TYPING 1. Slide (Modified Tube test) Uses whole blood suspended either in plasma or serum routinely done 2. Highly Protein Tube method Use whole blood; confirmatory incubate 10 minutes at 37C 3. Saline Tube RBC susp. In serum/plasma/saline, wash 3x with NSS to remove interfering factors, vaqluable in typing cells in (+) direct Coombs Test due to auto Ab Method of choice; disadvantage: long incubation Rh viewbox Maintain temperature at 45-50C for rapid warming of SX at 37C for rapid reaction, surface temperature viewbox 37C

(+) Coombs test Presence of autoantibodies E.g. hemolytic anemia

 REAGENTS TO DETECT D 1. Saline reactive anti-D Low protein base reagent; IgM =present Testing RC coaled with IgG Ab Cant use in testing Du or weak Du Prolong incubation is required, limited availability, cost of production 2. Chemically Modified IgG Sera Low protein base In slide and tube method Increase molecular weight pretention; enhance agglutination 3. Modified tube anti-D High protein base reagent contains IgG anti-D, increase concentration of protein, presence of macro moleculers additives (dextran and polyvinyl) Disadvantage causes false (+) result = increase concentration of bovine potentiator (22-30%) enhance medium LEWIS BLOOD GROUP SYSTEM Antigens: L ,Le
a b

Only blood group that is not manufacture by RC Antibodies: anti- Lea They are not synthesize and not incorporated in red blood cell membrane structure Antibodies: anti- Leb System, rather than blood group because Lewis Ag are found primarily in the body fluids and plasma Manufactured by tissue cells and secreted in body fluids and plasma and Ag adsorbed onto RBC membrane

Antigen production is dependent on inheritance 1. Le genes Required for the presence of Lea on RBC and saliva Le gene + H gene required for the presence of Leb on RBC Lea and Leb in saliva

2. Se genes Se gene and H gene- required for the presence of ABH antigens RBC and in saliva H gene without Se gene required for the presence of ABH antigens = RBC but not in saliva 3. Hh genes In the absence of H gene (hh) no ABH RBC and in saliva not contributory to H absence Le gene Produce enzyme -4-L-fucose
Lea

catalyzes transfer of fucose glucosamine where

Type I chain precursor substance in C4 of N-acetylglucosamine subterminal -4--fucosyl transferase transfer C appear in body fluids, secretions, red cells
b

Le

Leb gene not required in production of Leb Ag because it is dependent on the inheritance of H gene and Le gene The presence of H gene and Lewis gene + addition of fucose at 4C of nacetylglucosamine result in active Leb structure because H gene transfer fucose at 2C of D galactose Appear in body fluid and secretion, RBC if youre a secretor Enzymes that could enhance the reactivity: 1. Papain 2. Trypsin 3. Bromelin 4. Ficin Occur frequently in the sera of pregnant women

 Phenotype 1. Le (a- b+) Most common Secretors, LeABH ,Lea and Leb 2. Le (a+ b-) Non-secretor who inherit Le genes LeH and Leb 3. Le (a- b-) Non-secretors, never secrete Lea and Leb, but can secrete ABH if secretor gene is present/absent Glycoprotein Secretion Glycolipid RBC

H Precursor Substance Le Lea PS Hh

Se sese

Le (a-b+) Le (a+b-) Le (a+b-) Le (a+b-)

Se sese

Type 1 chain Le (a-b-) Le (a-b-) Le (a-b-) Le (a-b-)

Le (non-secretor)

PS

Se sese

hh

Se sese

 BLOOD GROUP SUSBSTANCES Genes H, Se, A, Le H, Se, OO, Le H, Se, A, Lele H, sese, A, Le hh, Se, A, Le M Secretions H, A, Lea, Leb H, Lea, Leb H, A Lea Lea Oh Erythrocytes H, A, Leb H, Lea, Leb H, A H, A, Lea Lea

Le Ab IgM (naturally occurring) Capable of bunding complement Enhance by enzymes Le substances in secretion can neutralize Le Ab Encountered in pregnant women Not considered significant in transfusion medicine MNSs BLOOD ROUP SYSTEM MNSs Have glycoprotein at chromosome 4 Well developed at birth Removed/destroyed by penetrating RBC with enzymes Anti-M and Anti-N (1927) K. Landsteiner and Levine S 1947 Walsh and Montgomery s 1951 U 1953 Weiner Ab of high frequency Means universal

Amino acids MN glycine serine

Found in glycoprotein A (shallow glycoprotein)

M 5, 4, 3, 2, 1 N 5, 4, 3, 2, 1

Glutamic Acid S,s,U Glycoprotein B, S,s shallow glycoprotein Differ in 2 aa at position 1 and position 5 M,N Phenotype M+NM+NM-N+ Genotype MM MN NN

leucine

Ss Phenotype S+sS+s+ S-s+ S-s-U-

Genotype SS Ss Ss SuSu

Anti-M
Naturally occurring (IgM) Cold reactive Saline agglutinins Dont bind complement Dont react with enzyme treated cells React best at pH 6.5 Rarely cause HDN and HTR

 Present in multiparous women Not clinically significant unless they react at 37C Demonstrate dosage Anti-N Naturally occurring (IgM) Cold reactive Saline agglutinins Dont bind complement Found in patient with renal failure undergoing dialysis treatment Doesnt cause HDN and HTR Anti-U Rare IgG React within AHG test Enhance with enzyme treatment Implicated in HTR and HDN Anti-S; Anti-s Implicated in HTR and HDN IgG react at 37C and the AHG phase or bind complement, reactive at 37C May or may not enhance by enzyme M lectins Japanese turnip Ibares amara Ibares umbellate Ibares semserinens Maclura amantiaea (made from asage orange) N lectins Bauhinia vareigata Bauhinia purpura Bauhinia candicans Bauhinia bionatiana Vicia graminea

 KELL BLOOD GROUP SYSTEM These are found in red cells but the Ag are not found in granulocyte, platelets, monocyte Well develop at birth They can be detect fetal RC as 10 weeks Can destroy by enzyme Kell rank 2nd in immunogenicity Kx gene Precursor substance If absent in RC leads to Mcleod Phenotype Mcleod Phenotype Abnormal RC morphology Smear: different shapes of RBC with pertrusions (acanthocytes) Result to chronic hemolytic anemia Reticulocytosis, splenomegaly, bilirubinemia, reduce haptoglobin levels Associated with sex linked chronic granulomatuous disease (hetero group of disease inability of phagocyte to ingest recurrent bacterial infection Kell antibody (Anti-Kell) IgG It is reactive to AHG phase Implicated to HDN, severe HTR Naturally occurring Associated with bacterial infection Organisms implicated with IgM anti-Kell Enterococcus faecium Morganella morgani and M. coli Mycobacteria Campylobacter jejunum DUFFY BLOOD GROUP SYSTEM Ags: Fya,Fyb produced/encoded by codominant alleles/genes Fya Identified in 1950 in point with hemophilia (Mr. Duffy) He have may blood transfusion

Fyb Described in 1951 Duffy Ag Protein in nature Identified in fetal RC as early as 6 weeks They are well developed at birth Considered as the Ag of the RC Are not found in point, monocytes, lymphocytes, granulocytes Can be identified in other body tissue, thyroid, thymus, lungs, skin endothelium, kidney cells Antibodies: Anti-Fya, Anti-Fyb Phenotypes: 1. Fy(a+ b+) Common in Caucasians (susceptible to malarial parasite) P. vivax 2. Fy(a b ) Marker in Africans and black race (resistant to malarial parasite) P.knowlesi 3. Fy(a b ) Chinese 4. Fy(a- b+) White Fy6 Receptor for invasion for Plasmodium vivax Fy3 Receptor for invasion for Plasmodium knowlesi Antibodies IgG React optimally at AHG phase Implicated in HDN and delayed HTR React with enzyme treated RBC
+ -

 KIDD BLOOD GROUP SYSTEM Simplest system Antigens: Jka and Jkb Antibodies: Anti- Jka Delayed hemolytic transfusion reaction Anti-Jkb Suffered a transfusion reaction (patients serum)

Well develop Not altered by enzymes Ag are not strong immunogens Not found in platelets, monocytes, granulocytes, lymphocytes if youre performing immunoflourescent assay Ab: have notorious reputation because it is destructive Demonstrate dosage effect IgG and anti-globulin reactive Enhance by enzymes Cause HDN Reagents: LISS- low ionic stress solution shorten incubation period PIG- polyethylene glycol mild HDN, delayed reaction

LUTHERAN 20 Antigens Not clinically significant

Lu genes Linked to secretor gene Found in Chromosome 19 along with Le, LW, S, H and C 3

Antigens: Lua 1945, serum of a point with LE low frequency Lub high frequency Antibodies: Anti- Lua Anti- Lub Recognized in 1956 by Cutbush and Chamain Ags Produced by allele Poorly developed at birth Can be detected on fetal RC as early as 10-12 weeks gestation Strength of Ag increase until 15 years of age Anti- Lu
a

blood with low frequency Ag

may naturally occurring saline agglutinin react at room temperature Anti- Lub IgG Reactive at AHG Associated with human expressive to erythrocyte Ag thru transfusion and pregnancy Ii BLOOD GROUP SYSTEM Dereived from individuality Similar to ABH and Le Ags Both I and I depend on age I- increase in RC of adults but have decrease and of i i- increase in infants: decrease amount of I Adult i cant be convert to I

Benign Anti-I Weak naturally occurring Saline reactive Autoagglutinin (IgM) Detectable only on 4C Seen in point with mycoplasma pneumonia infection Pathologic Anti-I Potent cold agglutinin T titer activity Antithetical Anti-i IgM agglutinin Reacts optimally at 4C Seen in point with IM, myeloid leukemia, alcoholic cirrhosis P BLOOD GROUP SYSTEM Related to ABO, Ii, and Le blood group system All of the Ag share a common precursor substance Ceramide dihexose or lactosyl ceramide Antigens: P P1 Expression is vatiable Poorly developed at birth Found in fetal cells as early as 12 weeks and weakened gestational age Pk Mechanism of expression of selected P blood group system Antigen Lactosylceramide Trihexosyl Ceramide (Pk Antigen) P gene Globuside (P Antigen) LKE

Sialosyl paragloboside (P Ag) Paragloboside PI gene PI Ag

Anti- PI Naturally occurring IgM in the sera of Pz individual Weak, Cold agglutinins (saline) Found in hydatid cyst fluid

Anti-P Naturally occurring alloantibody in the sera of all Pk individual Associated with PCH (Paroxysmal Cold Hemoglobinuria) hemolysis dual reaction 4 fixed complement 37 - lysis 2 Pathway: 1. Synthesis lead to globoside with P ag present in almost all erythrocytes 2. Produce paragloboside from P1 Ag Only P1 Ag is derived from the same precursor substance as the ABH Ag Sisalosyl Paragloboside Recognized by P Ag With sialic acid (differ from paragloboside residue) Lactosyl ceramide Altered by several sugar Addition of galactose in -1,4 linkage Cerenude dihexose Trihexosyl ceremide + sugar Anti- PP1Pk Produce by all P induce Increase frequency Ag IgM Bind complement Demonstrate in-vitro hemolysis Cause severe HTR, HDN Associated with spontaneous abortion in early pregnancy (Anti-Tja) trihexosyl ceramide globuside (carrying P angtigen) autoAb are IgG biphasic

In humans loboside is the precursor substitute for the Ag

Anti-P Naturally occurring alloantibody in the sera of all Pk individual Associated with PCH (Paroxysmal Cold Hemaglobinuria) hemolysis dual reaction 4 - fixed complement 37 - lysis 2 pathways: 1. Systhesis lead to globoside with P ag present in almost all erythrocytes 2. Module paragloboside from P1 Ag Only P1 Ag is derived from the same precursor substance as the ABH Ag Anti-PP1Pk Produce by all P individual Increase frequency Ag IgM Bind complement Demonstrate in-vitro hemolysis Cause severe HTR, HDN Associated with spontaneous abortion in early pregnancy Sialosyl Paragloboside Recognized as 10 Ag With sialic acid (differ from paragloboside residue) Lactosyl ceramide Altered by several sugar Addition of galactose in -1,4 linkage Ceramide dihexose Trihexosyl ceramide + sugar trihexosyl ceramide globoside (carrying P Ag) (Anti-Tja) AutoAb are IgG biphasic

In humans, loboside is the precursor substance for the Ag

DIEGO BLOOD GROUP SYSTEM Antigens: Dia, Dib, Wra ,Wrb Wda, Rba, and WARR low incidence antigens

High incidence Antigens: Low incidence Antigens: Dia

Dib, Wrb Dia, Wra

Codominant alleles on chromosome 17 Anthropologic marker for Mongolian Ancestry Anti-Dia, Dib, Wra Implicated to cause severe HDN CARTWRIGHT BLOOD GROUP SYSTEM Antigens: 1. Yta High incidence Ag; not well developed at birth , strongly immunogenic 2. Ytb a low incidence Ag; well developed at birth, poor immunogen Ab are IgG, reactive in indirect AHG, associated with transfusion reaction but not in HDN SCIANNA BLOOD GROUP SYSTEM Antigens: Sc1 A very high incidence Ag originally called Sm Sc2 A very low incidence Ag initially referred to as Bu2 Sc3 A high frequency Ag and present on RC carrying the Sc1 and or Sc2 Ags

Anti- Sc1 Binding complement Implicated with HDR Anti- Sc2 Implicated with mild HDN Dont bond with complement Implicated with HDR

 DOMBROCK BLOOD GROUP SYSTEM Common in American Indians Antigens: Doa, Dob Gya1 (Gregory) Hy (Holley) Joa (Joseph) Anti- Doa and Anti-Dob Implement encountered Reactive with AHG phrase and enzyme LISS enhancement Anti- Dob Implicated with HTR COLTON BLOOD GROUP SYSTEM Antigens: Coa,Cob,Co3 (formerly Coab) Ab: rare, causing HDN and HTR, detected by AHG phase CHIDO/RODGERS BLOOD GROUP 6 Chido Ags: CH1 CH2 CH3 CH4 CH5 CH6 2 Rodgers AGs RG1 RG2 WH HTLA (High Titer Low Avidity Antibodies) Exhibit reactivity at increase dilution of serum strength of agglutination is weak at any dilution

 CHROMER BLOOD GROUP SYSTEM Carried on the decay accelerating faster and Ags are distributed in body fluids, RC, WC, pt, placental tissue Antigens: 1. Cra 2. Jra 3. Dra 4. Esa 5. IFC 6. UMC 7. Wesb 8. Tcb 9. Tca 10. Wes Anti-cr Rare, rated in Black individuals Associated with mild HTR KNOPS BLOOD GROUP SYSTEM Antigens: 1. Kna 2. Knb 3. McCa (McCoy) 4. SIa 5. YKa (Yok2) Antibodies: IgG, RC stimulated (thru blood transfusion, pregnancy) Reacting weakly in indirect AHG Knops Ag are located on complement receptor (CR1) with expression being depressed by the In (Lu gene + autoimmune diseases) (Indian Lutheran)
a

High incidence

Low incidence

 INDIAN BLOOD GROUP SYSTEM Antigens: Inherited on codominant alleles in Chromosome 11 Ina More prevalent in Arabs and Iranians Inb Bg-white cell Antigens: Bennett Goodspeed Destroyed by treatment with chloroquine + glycine-HCl/EDTA solution 1. Bga 2. Bgb 3. Bgc Bg Abs: IgG, reacting weakly, variably in indirect AHG test GERBICH BLOOD GROUP SYSTEM A Complex system composed of high incidence antigens: chromosome 2 1. Ge2 2. Ge3 3. Ge4 Low incidence antigens 1. Wb 2. Lsa 3. Ana 4. Dha Antigens are carried on glycoprhorin C (GPC) + glycophorin D (GPD) GPC carries Ge3 and Ge4 GPD carries Ge2 and Ge3 Ge2 , Ge4Wb, Lsa, Ana, Dha destroyed by papain and ficin Ge4 resist protease treatment Anti-Ge IgG, may have IgM component Clinically significant Rare, cause HDN correspond with Class I HLA antigens B7,B17,A28

 Variable reaction Xg BLOOD OPPURTUNISTIC SYSTEM One of the most unique blood group Only 1 Ag is recognized Xga recessive Phenotype: 1. Xg (a-) 2. Xg (a+) Genotype: XgXg Xga Xga Xga Xg Anti-Xga A uncommon Ab which reacts at room temperature (37C) react Not implicated with HDN, HTR SID BLOOD GROUP SYSTEM Antigen: Sda Anti Sda Weakly reactive Some react at room temperature or 37C Reactive in AHG phase Can be demons with enzymes treated RC Reaction enhance with Liss Not associated with HDN and HTR Sda Ag Inherited as autosomal dominant ANTIGLOBULINS OF COOMBS TEST Must important serologic procedure in BB Used to detect incomplete Ab or non-agglutinating Ab Provide as a tool for the detection of 7s or IgG which either dont react in saline as increase protein media common in female rather than males sex linked

Complete Ab/Saline Ab/Bivalent Has 2 receptor sites at each end up Ab molecule (albumin, monovalent, divalent) Incomplete Ab (albumin, monovalent) 1 receptor Must bridge by protein colloid or Anti-Human globulin reagent AHG=anti-IgG +RC sensitized Principle: AHG Ab induce agglutination of erythrocytes coated with globulin

+AHG polyspecific

Anti-IgG

RC AHG Bridge antibody molecules A. DIRECT AHG In-vivo sensitization Not routinely done

no agglutination

Useful in the investigation of transfusion reaction Dx of autoimmune hemolytic anemia Dx of HDN Detection of RC sensitization caused by drugs (penicillin, methyldopha, cephalotin) (+) result = indicates RC coated with IgG or complement B. INDIRECT AHG In-vitro sensitization Serum + RC Ab Ag incubate 37C wash with NSS (3X) + AHG reagent allows the attachment of IgG on RC Detection of Du, crossmatching, RC Ag phenotyping, investigation of transfusion reaction Detection and identification of unexpected Ab Preparation: Immunizing guinea pig/rabbits with Human globulins

 Factors affecting AHG test: 1. Washing phase = inadequate washing = false (-) result in neutralization AHG result 2. Control used Group O cells Over = erroneous result 3. Manner of reading SENSITIZATION PHASE 1. Temperture = 37C demonstrate IgG to RC 2. Incubation = 15-60 minutes 3. Ratio of serum to cells = increase serum =increase sensitivity minimum 40:1 (2gtts seum and 1 gtt 3% RC) 133:1 (4gtts serum + 1gtt 3% RC 4. Reaction medium Use enhancement media (22% bovine albumin, LISS, PEC) Increase dielectric constant (IgG Ab demonstrated) LISS reduce incubation (20 or 10 minutes) PEG additive; increase Ab uptake, removes H20, Ab becomes concentrated Polyspecific Reagent Mixture of Ab (anti-IgG, anti-complement-C3D) Monospecific 1 Ab COMPATIBILITY OR CROSS-MATCHING TEST Most important procedure in BB It is a series of procedure designed to ensure safety of blood for transfusion\ Performed to prevent transfusion reaction due to Ab Ensure maximum benefits to recipient 2 forms: 1. Major cross-match Most important part Consist of mixing of patient serum and donors RC (PSDR) Detect presence of Ab in patient serum that may damage donor RC 2. Minor cross-match Mixing the donor serum and patient RC (DSPR)

 Detect Ab in the donors serum that may react with patient RC Obsolete donor units are screened already Specimen: fresh; non-inactive, <48 hours old Serum rather than plasma Clotting (fibrin) Activate complement some Ab may not be Detected Not intravenous line (blood) avoid contamination with material confusing result Remedy: step the line for 10 minutes, discard the 1 st tube Store the specimen for 7 days in refrigerator (1-6C) METHODS: 1. Saline Cross-matching Routinely done Disadvantage: long incubation period (60 minutes) Principle: patient serum and RC suspended in saline-hemolysis and agglutination @22C detect ABO incompatibility, cold agglutinins, autoAb, detect some Anti-M,N,S Detect saline reacting Ab (anti-Lu, anti-Le, anti-P) @37C detect most anti-Rh Ab, some anti-MNS, anti-kell, anti-Le 2. High Protein Cross-matching PS + DR + Bovine albumin hemolysis, agglutination Detect Rh Ab, anti-Fya, anti-Kell, @37C detect most anti-Rh Ab, some anti-MNS, some anti-Le Ab 3. Antiglobulin Detect unexpected Ab such as anti-kell, anti-Fy, anti-Kidd PS + DR + AHG hemolysis and agglutination 4. Enzyme Technique As a back-up test for indirect Coombs test Used in investigation of delayed Transfusion reaction Ab identification rather than Ab detection of irregular Ab Papain pauypaw (Carica papaya) Ficin from fig tree (Ficus carica) Bromelin extract from pineapple (Ananan sativus)

Trypsin hogs stomach 5. Broad Spectrum Compatibility Testing Most important method; method of choice 3 phases: 1. Protein (room temperature phase) Detect: ABO incompatibility Incompatibility due to cold Ab Prozoning Ab (detected in serum mixture of immediate centrifugation) 2. Thermophase Detect: incompatibility due to presence decreased titer Certain Rh Ab (Anti-C, Anti-E, Anti-D) 3. Antiglobulin phase Detect: anti-Fy, anti-kidd, anti-Kell Ab present in acquired hemolytic anemia Ab in Rh system which react in AG phase (3rd order or cryptoagglutinoid)

PSDR PS 5% DRCS DS 5% 22% PRCS Bovine albumin 2gtts 2gtts -

DSPR 2gtts 2gtts 2gtts 2gtts 2gtts 2gtts 2gtts 2gtts

Incubate for 10 minutes (37C), centrifuge (1 minute) If theres agglutination repeat from the start If no agglutination discard tube B and D Tube A and C wash with NSS (3X) (-) autocontrol caused by specific Ab (+) crossmatch

(-) autocontrol patient dont have specific Ab (+) crossmatch

E.g. PSDR P=O D=A P=AB D A Anti-A Anti-B A DSPR Anti-B none

incompatible A

Compatible Anti-B A 1B

None

compatible

Incompatible

P= O Rh-(with pervious immunization) D= O Rh+ PSDR Anti-D D DSPR None none

incompatible

Compatible

Type O packed RC Rh+ (in case of emergency : unknown blood group) In babies= mothers serum and donors serum AHG test= test for unexpected/irregular Antibody use at least 10 group O Screening poded group O cells Panel cell test= for identification of unexpected antibody Abbreviated cross-match/immediate spin saline technique 2gtts PS, DR Centrifuge, observe for agglutination If patient has no known irregular antibody Continue with incubation and AHG test after release

 MAIN GOALS: To prevent physical changes detrimental to the constituent Minimize bacterial proliferation Maintain viability and function of each relevant RC constituents RBC viability Measure of in vivo RC survival following transfusion Reflected in ATP Hemoglobin function Depend on 2,3, DPG ANTIGOAGULANTS 1. CPD - 21 days stored at 1-6C 2. ACD 21 days stored at 1-6C 3. CP2D - 21 days stored at 1-6C 4. CPDA- 35 days stored at 1-6C Citrate Binds Ca+2 Preventing activation of coagulation Dextrose Provide the energy for the RC Adenine Provide substrate from which RC synthesize Phosphate Provide RC to maintain 2,3,DPG Slow glycolic activity 5. Heparin- 48 hours use it for open-heart surgery 6. EDTA- preparation of platelet concentrate 7. Ion-Exchange Cesin- 24 hours copural and exchange transfusion Adisol (As-1) 42 days, coupled with CPD Optisol (As-5) - 42 days, coupled with CPD Nutricel (As-3) - 42 days, coupled with CPD Nutricel (As-2)

Blood Additive Consist of saline; adenine, manitol (Red cell stabilizing agent) Increase concentration of dextrose Rejuvination Solution Can regenerate both ATP and 2,3-DPG level Consist of PIGPA (Phosphate, Inosine. Glocose, Pyruvate, Adenine) Blood Substitute use Hb base O2-cause (recombinant Hb, chemically modified Hb, stroma free-Hb solution, encapsulated Hb, perfuro chemical) in order to keep erythrocyte in frozen state- acid cryoprotective cysts Cryoprotective agent prevents severe inquiry by altering the tonicity of the cell 1. intracellular (penetrating) use glycerol or dimethyl sulfoxide 2. extracellular (non-penetrating) HES (Hydroxy Ethyl Starch) 3 METHOD OF FREEZING RC 1. High Glycerol Slow Freezing (HGSF) Use 40% glycerol, it is stored at -65C in a container made of liquid nitrogen freezer provided the temperature 2. Low Glycerol Rapid Freezing (LGRF) Use 20% glycerol stored at -120C in a container made of polypolytene liquid nitrogen freezer provided the temperature 3. Agglomeration Uses glycerol increase concentration of sugar stored at -65C, the container is made of polyvinyl ClDeglycerolization Removal of glycerol HGSF Wash RC in decrease of NaCl + 12, 1.6, 0.9% LGSF Wash RC with 0.45 NaCl in 15% manitol + 0.9 NaCl

Agglomeration Wash RC with 50% dextrose + 5% fructose + 0.9% NaCl BLOOD COMPONENTS Products prepared form a single unit of whole blood, packed RBC Considered as drugs treating diseases Transplant ation, transfusion of RC have to survive I. Whole Blood Taken from donor, no need for a process Contain all cellular components of blood Enhance O2; increase blood volume Give to patient bleeding massively 1 unit of blood = increase hematocrit (3-5%); increase hemoglobin (1-1.5 gm/dL) Easiest to prepare WB Hard spin Plasma RBC Deep freeze (-18C) Thaw overnight FFP (37C) Hard spin: apparatus (refrigerated centrifuge) washing-machine like 5000-8000 rpm 5-8 minutes Soft spin: 2000-3 minutes Multiple bag mother bag and have satellite bag II. RBC/Packed RBC Prepared by overnight sedimentation/centrifugation (inside biological refrigerator) Remove 250 mL plasma (by plasma extraction) Clinically significant: patient with severe chronic anemia Increase O2 carrying capacity of RC III. Granulocyte Concentrate Leukocyte concentrate Prepared by phoresis (open sep.) transfuse within 2 hours product contained 1.0X1010 granulocytes of steroids and Hydroxyl Ethyl Starch (HES) is used Given to the donor within 24 hours stored at room temperature 22-24C without agitation Clinical individual: severe neutropenia, patient with fever (unresponsive to antibiotic therapy, myeloid hypoplasia of bone marrow IV. Leukocyte Poor RBC Products which 70% if original leukocyte removed and 80% erythrocyte remained

1. Inverted centrifugation (15-20 minutes) 2. Washed RC like the preparation of 5% RC suspension Washed RC: patient with autoimmune hemolytic anemia, PNH, transfusion reaction 3. Sedimentation Increase molecular weight polymers suck as dextrin, gelatin, hydroxyl ethyl starch 4. Filtration Specific leukocyte depleting filters, microaggregate filters with a size of 20-40nm) WB Soft spin (2000g-3 minutes)

Platelet rich plasma

PRBC

Hard spin

Platelet cone

Plasma

Deep freeze (-18C) FFP

Thaw overnight (1-6C)

Cryoprecipitate

cryosupernatant

Thaw at 37C

Liquid cryoprecipitate

liquid cryoprecipitate

a. Amount of blood to be drawn Donors wt (lb) X 450 mL = allowable amount (mL) 110 b. Amount of anticoagulant needed Allowable amount X14 = anticoagulant needed (mL) 100 c. Amount of anticoagulant to remove 63 mL anticoagulant (mL) = anticoagulant to removed (mL) 450 mL +- 45 mL + 63 mL anticoagulant +30 mL (pilot tube) e.g. Donors weight X 450 mL 110

= 90 X 450 110 =368.18 mL

=268.18 X 14 100 =51.54 63 mL anticoagulant =63 mL-51.54 =11.46 mL Blood donation: 6X per year 8weeks or 2 months interval TRANSFUSION REACTION Any adverse effect which result from a blood transfusion Procedure by incompatibility between a patient and a unit of donor blood Fatal reaction result in misidentification or clerical errors rather than technical errors Fatal Transfusion Reaction: (clerical errors) 1. Misidentification of the patient 2. Mislabeling of specimen 3. Errors in laboratory records 4. Mistakes in blood typing 5. Inaccurate Ab screening/crossmatching 2 major groups: 1. Acute/ Immediate Occur during/shortly after the infusion of blood production Observed within 48 hours of administration 2. Delayed Observed in 7-10 days

Allowable amount X 14 100

 Hemolytic Transfusion Reaction: Due to incompatibility between the recepients and donors unit blood Most fatal but the least common Caused by clerical errors Major Categories: 1. Intravascular hemolysis Hemolysis: red cell distruction occur in blood vessel rapid increase free hemoglobin in the circulation 2. Extravascular hemolysis Primary site of RC destruction is reticuloendothelial system Imcomplete of activation of complement Ab: Anti-E, Anti-c, Anti-Kell, Anti-Jka Classical Signs and Symptoms: Fever and chills, lumbar pain, pain in the lower back, pain in the arm that was used in transfusion Physical signs: Fever , hypotension, hemoglobinuria, breatheing/renal failure, uremia, death Immediate Non-hemolytic Transfusion Reaction 1. Febrile transfusion reaction Cause by anti-leukocyte Ab present in the patients plasma Most common Due to platelets, leukocytes various plasma constituents, bacterial pylogens Associated with patients with multiple platelet transfusion, non-specific leuko agglutinins, those HLA origin have been implicated Treatment: administration of anti-histamine, blood-component-leukocyte poor component Characterized by fever, with or without chills, rarely hypotension 2. Allergic Transfusion Reaction/Utecarial/Hives and Itching 2nd most common type of transfusion reaction Reaction between soluble materials in transfused units and Ab in patients circulation Treatment: anti-histamine Characterized by itching, local erythema, no fever 3. Anaphylactic Transfusion Reaction Patients have congenital IgA deficiency developed anti-IgA Anaphylactoid Transfusion Reaction Normal levels of IgA but a limited type of specific anti-IgA that reacts with the light chain of the donors IgA Fever is absent Stop the transfusion immediately, give 0.5/1000mL solution of epinephrine Corticosteroid severe reaction Amonophyllin

 Non-cadiogenic pulmonary edema NCPE TRALI Transfusion Related Acute Lung Inquiry PHR Pulmonary Hypersensitivity Reaction TACO Transfusion Associated Circulatory Overload Too fast transfusion of large volume of blood congestive heart failure or respiratory complication Use PBRC, administer blood slowly, rte: 200mL/hr in case of severe chronic anemia: 100 mL/hr Increase of bacterial growth = slow transfusion reaction Diseases: 1. Malaria Transmitted by using PWB, platelets, and allow composition with RBC 2. Hepatitis A Oral-fecal route Infection; short incubation Hepatitis B Transmitted by transfusion: long incubation Classic example of virus transmitted thru blood transfusion 3. Syphilis Trponema pallidum Not occur in serrous transfusion Dont survive at 4C in 72 hours 4. IM Epstein virus 5. Cytomegalo virus IM 6. Hepatitis C Dx of exclusion because of absence of serologic markers and viral origin 7. Factor 8 and Factor 9 Commercially prepared, lyophilized product prepared from pooled plasma by Cohn Ethanol Fractionation Treatment of Hemophilia A (F8) Treatment of Hemophilia B (Fa) 8. AIDS BLOOD COMPONENTS (continuation) V. Fresh Frozen plasma Prepared from whole blood by centrifugation and separating alting about 200-260 plasma within 8 hours of collection to maintain labile coagulation Frozen at -18C

1 mL plasma = 100% or 1 unit coagulation activity Before using thaw at 37C with constant agitation, transfuse within 24 hours stored at 1-6C Single Donor Plasma Liquid Separated from WB after 24 hours stored 4C (used in shock and burns) Single Donor Frozen Plasma Prepared within 24 hours collection stored at -18C Used in multiple coagulation deficiency treatment VI. Cryoprecipitate Prepared from whole blood Contains 80 units of FxIIII:c, about 50% von Willebrand factor 200 mg fibrinogen, 25% FxIII, 25% fibrinogen Slowly thawing overnight of FFP at 1-6C leaving small amount of white decrease centrifuge leave 10-15 mL plasma on the decrease refrozen at -18C When needed thaw at 37C used within 6 hours stored at 1-6C No. of bags required: Desired level factor of FvIII% X plasma volume (mL) 80 are units of FvIII in a single pooled plasma Cryosupernatant Plasma left after separation of fresh blood treatment of bleeding tendency other than hypofibrogenomia, hypobulemia, Hemophilia VII. Platelet concentrate From fresh whole blood, platelet rich plasma, patients RC, patients concentrate Products: patients concentrate, patients rich plasma Prepare: Random donor platelets: prepared from WB within 8 hours of collection by centrifugation (light spin) to produce u-rich plasma satellite bag (recentrifuge-hard spin) P. poor plasma/reaggregated cell button Store at room temperature, continuous aggregation 5.5X1010 pl Shell life: 5 days Single donor plateles: prepared by pheresis (open system) 24 hours shelf-life 3.0X1011 platelets Stored at room temperature with continous agitation used in treatment of thrombocytopenia and thrombocytopathy Corrected count increment: 1. Post trans Pre-trans X body surface (m2) Pl Pl ct area 11 # of pl transfused (10 ) (multiple of 1011)

# pl. transfused X 1011 =0.55 # pl. in each unit with concentration expressed in 1011 2. Percent recovery Post trans pl. ct Pre trans pl ct. X blood volume Total # of pl infused X 2/3 Refractory state Resistant to ordinary treatment Investigation of suspected Transfusion Reaction Specimen needed: a. Pretransfusion blood of recipient b. Post transfusion blood of recipient c. Blood from integral donor tubing implicated in reaction d. Post transfusion urine Immediate investigation: Examine for hemolysis (a,b,c) Repeat: ABO and Rh Direct AGT (a,b) Definitive Investigation: Repeat: cross-matching (a,b,c) Ab screening (a,b,c) Corroborative Investigation 1. Identification of unexpected Ab or incompatibility 2. Bacteriologic smear and culture Optional Investigation 1. Haptoglobin (a,b) 2. Bilirubin (b) 3. Creatinine 4. DAGT Calculating dosage for and of FxIII required 1. Weight (kg) X 70 mL/kg = blood volume (mL) 2. Blood volume (mL) (1.0-Hct) = plasma volume (mL) 3. Plasma volume (mL) X desired factor VIII Level u/mL = initial Factor VIII Level u/mL = unit required e.g. to raise FvIII level to 50% in a 70 kg patient with Hct of 0.04 (40%) and have initial factor level of O%. Compute the amount of FvIII required? 70 X 70 = 4,900 mL 4900 X (0.6) = 2,940 2940 X 0.5 0 = 1470

Neocytes Younger RBC prepared by differential centrifugation Treatment of youg patient with thalassemia (multiple transfusion) Prevent Fe+3 overload FvIII and FIx concentrate Prepared by Con-ETDH conc Hemapheresis Separate/remove Procedure: separate different components, 1 or more components retained, ether constituents returned to the individual Used to obtain components intended for transfusion, use to remove pathologic elements: c, dissolved plasma factors circulating (leukocytes, RC, pl) Erythropheresis Remove RC, remain plasma, leukocyte, pl METHODS 1. IFC (Intermitent Flow Centrifugation) Performed in cycles, need only 1 venipuncture site Products: RBC, WBC, platelets, plasma 2. CFC (Continuous Flow Centrifugation) Withdraw, process, return the blood to the individual simultaneously 2 venipuncture site is necessary Machines: Membrane Filtration Technology Use with specific pore plasma collection Plasma collection Advantages: collection of free product Ability to selectively Fluids: Anticoagulants ACD Acc. heparin Normal saline to heep the line open; maintain open Hydroxyl ethyl starch sedimenting reagent especially granulocyte collection-better separation between RBC and WBC FFP contains all constituents of the removed plasma, optimal replacement of fluid Things to remember: Interval between Apheresis procedure at least 24 hours

 Amount of RC used should not be performed more than 2X in 1 week or 24X in 1 year unless approved by BB personnel If the donors RBC cant be reinfuss during a procedure 1 and if a patient donates a unit of WB for weeks should elapse before subsequent to cytopheresis procedure unless the Hb required is met, the donor is found acceptable by a BB physician Extract porporial blood volume (the volume out of the donor) should not exceed 15% of the donors estimated total blood volume Pheresis Best accepted collected HLA compatible donor preferable nearest relative ABO, rh, Hb, Hct, HbSAg Autologous Transfusion Self-donation, donation of blood by a intended recipient, the patients own blood is safest Surgical procedure Importance 1. Eliminate the possibility of transfusion reaction 2. Prevents transmission of infectious diseases 3. Norisk of alloimmunization (RBC, platelets, WBC, plasma proteins) 4. Phlebotomy process stimulates BM to increase production Different types: 1. Pre-deposit Get blood store until it is needed (label for autologous transfusion 2. Intraoperative Hemodilution Collection of 1 or 2 units of blood from the patient just before surgical procedure, replace the blood volume by crystalloid/colloid solution at the end of the surgery, blood units are reifuse to the patient. 3. Intraoperative Salvage Blood is salvage/aspirated from the surgical field and infuse, during or after surgical procedure 4. Postoperative Salvage blood is collected into a sterile plastic lines, field with blood filter with or without prior washing, return blood to patient Criteria: No age limit, no weight limit Hgb 11dL Hct 33% Frequency should not be more frequent not more than every 3 days final donation should be Phlebotomy withdrawal of blood

 Hemolytic Disease of the New born Isoimmune disease, characterize by RC destruction during fetal life and cause by fetomaternal blood group incompatibility Clinical manifestation: anemia, jaundice, generalized edema (hydrops fetalis) enlargement of the liver, spleen, bilirubin and encephalopathy (untreated) kernicterus 2 Main types: 1. Incompatibility (ABO) Hemolysis of fetal RC brought about by maternal Anti-A/Anti-B IgG alloantibody (peripheral smear = spherocytosis) Mild disorder because: Ab is neutralize by solution A and solution B substance in fetal circulation Fewer A and B Ag sites in the newborn Weaker Ag strength in the newborn (A and B Ag) Competition of Anti-A and Anti-B between tissue and erythrocyte Sensitization occur when mother is pregnant (have immunization) 2. Rh incompatibility Mother=RhFather=Rh+ infant=Rh+ Hemolysis of Rh+ fetal RC brought by maternal Anti-D (IgG Ab) as the placenta separate from the uterus (peripheral smear: reticulocytosis) RC from fetus foreign mother- sp. Ab Different Laboratory Diagnosis: 1. Aminocentesis 2. Intrauterine transfusion 3. Untrasound 4. Cordocentesis Percutaneous Umbilical Blood Sampling Needle to umbilical vessel aspirate blood sx blood type, Hb, Hct, direct antiglobulin test Treatment: Exchange transfusion Remove continuous blood from the infant (FB<5 or 7 days old) Treatment of anemia and hyperbilibinemia Bilirubin cause damage in the brain Supply erythrocyte to correct anemia Remove erythrocyte coated with Ab Remove free-Ab which causes additional hemolysis Prevention: 1. Spacing of pregnancy (4 years) 2. Administration immunoglobulin (Rhogum) Rh- mother within 72 hours after delivery Commercially available

Criteria for administration of Rhogun: 1. Patient: Rho D- Du2. Maternal serum doesnt contain Anti-D 3. Infant: Rho D+ or Du+ All IgG: should evaluate for fetomaternal bleeds exceeding 30 mL Techniques demonstrated fetal RC: 1. Kleihauer Betke Acid Elution Use to investigate the incidence Trans-placental hemorrhage before onset of the labor Fetal RC resistant to elution in an acid medium Hb A = susceptible to acid medium Peripheral smear = stain at fetal RC 2. Rosette Formation Determine the volume fetal RC and # of Rh Ig vials needed 3. ELISA 4. Flow cytometry detect small volume of Rh+ # fetal RC (%) X 50 = volume of fetal RC Volume of fetal RC = # Rh Ig vials needed 30 e.g. 3% X 50 = 150 mL 150 mL = 5 vials 30 If the # of the right decimal point <5 roundown, add 1 vial >5 round up to the next # add 1 vial e.g. 2.4 = 2+1 = 3 3.9 = 4+1 = 5 3.5 = 4+1 = 5 2.1 = 2+1 = 3