(pt 1) exam #2 - immunohematology (cls 544)

abo discrepancies, rh system

categories of ABO discrepancies

• Group I Discrepancies

• Group II Discrepancies

• Group III Discrepancies

• Group IV Discrepancies

ABO discrepancies (general)

• Unexpected reactions in the forward and/or reverse typing due to:

• Can appear as extra positive or weak/missing reactions

• Must be resolved prior to reporting a patient or donor's ABO group

main causes of ABO discrepancies (generally; 3)

• Problems with the patients' serum/plasma

  • Missing or weak reaction in reverse typing

  • Unexpected positive result in reverse typing

• Problems with the patients' red blood cells

  • Missing or weak reaction in forward typing

  • Unexpected positive result in forward typing

  • Mixed field

• Problems with both the patient serum and RBCs

group I discrepancies

• Associated with unexpected reactions in the reverse typing due to weakly reacting or missing antibodies

  • Depressed antibody production

  • No ABO antibodies

• Discrepant results in reverse typing more common than in forward typing

• Common population: newborns, elderly patient, immunocompromised, etc

interpretation of 39.2 F (4 C) reverse ABO group results & action:

• reverse type: matches forward type results

• group O cells: 0

• autocontrol: 0

interpret the ABO group as normal; no discrepancy present

interpretation of 39.2 F (4 C) reverse ABO group results & action:

• reverse type: does not match forward type

• group O cells: any result

• autocontrol: any result

do NOT interpret ABO group; investigate possible cause of discrepancy

interpretation of 39.2 F (4 C) reverse ABO group results & action:

• reverse type: any result

• group O cells: +

• autocontrol: any result

cold-reactive antibody other than ABO present

• identify the cold-reactive antibody and repeat ABO-reverse group using cells that are negative for the corresponding antigen

interpretation of 39.2 F (4 C) reverse ABO group results & action:

• reverse type: any result

• group O cells: any result

• autocontrol: +

cold agglutinin (antibodies that react at RT; aka “cold-reacting”)

• do NOT interpret ABO group

group II discrepancies

• Associated with unexpected reaction in the forward typing due to weakly reacting or missing antigens (the least common)

  • ABO subgroups or weak ABO subgroups

  • Diseases that alter the ABO antigen expression

  • Excessive soluble substances

• Enhance weakly reacting antigens with room or colder temperature incubation

• Pretreatment of RBCs with enzymes

(group II) causes of unexpected positive reactions in forward type

• Substances in plasma if testing done using unwashed cells

• Acquired B—bacterial infection

• More than one ABO group: mixed field agglutination

  • True chimerism or mosaicism

  • RBCs transfusion

  • Bone marrow/stem cell transplantation

  • Fetal-maternal bleeding

• Unexpected other antibodies

  • Anti-B reagent

    • Person makes ABYs that recognize yellow dye

group III discrepancies

• Protein or plasma abnormalities result in rouleaux formation

• Rouleaux is described as the non-specific agglutination of RBCs

  • Diseases

  • Wharton's jelly

• Saline replacement technique

when to suspect rouleaux?

if all transfusion medicine tests that use the patient’s plasma are positive

• ex: in tube testing: reverse ABO group, immediate spin crossmatch but not the indirect antiglobulin (IAT) phase of antibody screens or crossmatches

• and one or more of the following:

  • pt has multiple myeloma or Waldenstrom’s macroglobuilnemia or other disease that results in high serum protein/abnormal serum proteins

  • agglutination looks like stack of coins microscopically

  • high MCHC

  • pt has receive a plasma volume expander that contains high MW substances (dextran)

group IV discrepancies

• Cold autoantibodies

• Unexpected ABO isoagglutinin

  • Tested with Dolichos biflorus to confirm the presence of an A subgroup

  • Cis-AB

    • Inheritance of both A and B genes from one parent on same (cis) chromosome 9

    • O gene inherited from the other parent

    • Offspring inherit 3 ABO genes instead of 2

• Unexpected non-ABO alloantibodies (Anti-M)

(resolution of ABO discrepancies) anti-A₁

to confirm the presence of anti-A₁ in an individual who has not been recently transfused, type RBCs with anti-A₁ lectin (see pic for testing algorithim)

plasma reaction patterns for identification of anti-A₁ & interpretation:

• A1 pos cells: +

• A1 neg cells: 0

• O cells: 0

interpretation: anti-A1 present

plasma reaction patterns for identification of anti-A₁ & interpretation:

• A1 pos cells: some or all +

• A1 neg cells: + or 0

• O cells: some or all +

interpretation: antibody other than anti-A1 alone or in combination with anti-A1

plasma reaction patterns for identification of anti-A₁ & interpretation:

• A1 pos cells: 0

• A1 neg cells: + or 0

• O cells: some or all +

interpretation: antibody other than anti-A1

plasma reaction patterns for identification of anti-A₁ & interpretation:

• A1 pos cells: 0

• A1 neg cells: 0

• O cells: 0

interpretation: likely an antibody to a low-incidence antigen

techniques to rule out ABO discrepancies

• Check for any clerical errors

• Check for any technical errors

• Confirm daily reagent QC results

• Repeat testing

resolution strategies for ABO discrepancies (general)

• Check manufacturer's instructions

• Run (or repeat) appropriate controls

• Follow your laboratory's standard operating procedure

• Blood components from donors with unresolved ABO discrepancies may be diverted for non-transfusion purposes

(ABO discrepancy examples) interpret the following:

• Anti-A: 4+ ; Anti-B: 0

• A1 cells: 0 ; B cells: 0

likely group A with missing anti-B

• alternate: group AB with missing B antigen

(ABO discrepancy examples) interpret the following:

• Anti-A: 1+ ; Anti-B: 4+

• A1 cells: 4+ ; B cells: 0

likely group B with extra reaction with anti-A

(ABO discrepancy examples) interpret the following:

• Anti-A: 1+ ; Anti-B: 4+

• A1 cells: 2+ ; B cells: 0

likely group B with an extra reaction with anti-A

• alternate: group AB with a weakly reactive A antigen and an extra reaction with A cells

(ABO discrepancy examples) interpret the following:

• Anti-A: 0 ; Anti-B: 4+

• A1 cells: 4+ ; B cells: 2+

likely group B with an extra reaction with B cells

(ABO discrepancy examples) interpret the following:

• Anti-A: 0 ; Anti-B: 0

• A1 cells: 0 ; B cells: 4+

likely group A with missing A antigen reaction

• alternate: group O with missing anti-A reaction

(ABO discrepancy examples) interpret the following:

• Anti-A: 0 ; Anti-B: 0

• A1 cells: 0 '; B cells: 0

likely group O with missing antibody reactions

• alternate: group AB with missing antigen reactions

(ABO discrepancy examples) interpret the following:

• Anti-A: 4+ ; Anti-B: 0

• A1 cells: 1+ ; B cells: 4+

likely group A with extra reaction with A cells

(ABO discrepancy examples) interpret the following:

• Anti-A: 4+ ; Anti-B: 4+

• A1 cells: 2+ ; B cells: 2+

likely group AB with extra reactions with A and B cells

• suspect possible rouleaux

(ABO discrepancy examples) interpret the following:

• Anti-A: 2+ ; Anti-B: 2+

• A1 cells: 4+ ; B cells: 4+

likely group O with extra reactions with anti-A and anti-B

(causes of ABO discrepancies) missing or weak reaction in forward type

• ABO subgroup

• diseases that alter the ABO antigen expression (leukemia, Hodgkin’s lymphoma)

• transfusion of transplantation

• excessive solution ABO antigens in pt/donor’s plasma

(causes of ABO discrepancies) missing or weak reactions in reverse type

• age (neonates/elderly)

• hypogammaglobulinemia (either primary disease or secondary to leukemia/lymphoma)

• agammaglobulinemia or other congenital immunodeficiencies

• patients on immunosuppressive therapy

• transplantation

• patients whose antibodies have been diluted by plasma exchange therapy

• ABO subgroup

(causes of ABO discrepancies) unexpected positive reaction in the forward group

• panagglutinating cells due to changes in red blood cell membrane or interference from plasma proteins

• Wharton’s jelly in cord samples

• acquired B antigen

• B(A) phenomenon

• transplantation

(causes of ABO discrepancies) unexpected positive reaction in reverse type

• ABO subgroup

• cold reactive auto/alloantibody

• antibody to reagent

• excess serum protein due to multiple myeloma, Waldenstrom’s, or other disease

• treatment with plasma expanders (dextran)

• passive ABO antibodies (e.g. transfusion of IVIG, maternal transfer of antibodies to child)

(causes of ABO discrepancies) mixed field agglutination in the forward group

• recent transfusion

• transplantation

• feto-maternal hemorrhage

• ABO subgroup, notably A3

• chimerism (twins/dispermy)

(causes of ABO discrepancies) different current ABO group from historic ABO group

• patient/sample ID error during collection

• patient is victim or perpetrator of identity theft

• sample mix up during testing

• pt has received an ABO nonidentical BMT

intro to Rh blood group system

• Rh blood group system is one the MOST important systems in transfusion medicine

  • Considered second to ABO blood group

  • Rh antigens are VERY immunogenic (are proteins)

• Clinically significant Rh antibodies are common in pregnancy and in blood transfusion recipients

• Produced in response to an incompatible transfusion or pregnancy

rh blood group system history (1939-1940)

• 1939: Levine and Stetson first described a hemolytic transfusion reaction case involving an OB patient

• 1940: Landsteiner and Wiener discovered an antibody made by rabbits and guinea pigs after injection with Rhesus monkey RBCs ; anti-Rh(D) discovered and named after the rhesus monkeys

  • For each case, there was a different Rh antibody responsible

    • Rh retained for the antibody produced by humans (anti-D)

    • Anti-LW for the Rh antibody produced by rabbits and guinea pigs

• Further research led to implication of Rh antibodies as the primary cause of HDFN and a significant cause of HTRs

rh blood group system history (1940s-today)

• By the mid-1940s, 5 antigens made up the Rh blood group system

• 1982: identification and isolation of Rh polypeptides

  • RhD and RhCE proteins

• 1986: Rh genetic expression controlled by two closely linked genes

  • RHD and RHCE genes located on chromosome 1

• Today: over 60 different specificities within the Rh blood group system

what chromosomes are the RHD and RHCE genes located on?

chromosome 1 (!!!)

biochemistry of Rh antigens

Rh specific antigens reside on transmembrane PROTEINS vs the carbohydrate antigens of ABH antigens

• Cross the RBC membrane 12 times, 6 small extracellular loops where the antigens are expressed on the surface

  • Maintain RBC structural integrity

  • Maybe act as molecular transporters

• Rh phenotypes controlled by two genes

  • RHD—encodes for D antigen

  • RHCE—encodes for Cc and Ee antigens

characteristics of Rh antigens

• Rh antigens are non-glycosylated proteins expressed on the RBCs

• Rh antigens are very immunogenic

  • Well developed at birth

  • D antigen is the most immunogenic of all Rh antigens

  • Remember: D > c > E > C > e

• D antigen causes immunization at least 50% of the time in cases where a Rh D-negative individual receives one unit of Rh D-positive RBCs

• Number of D antigen sites vary depending on the Rh phenotype

• C/c and E/e antigens, co-dominate alleles

• If gene present for antigen, it will be expressed on RBCs

order of immunogenicity of Rh antigens

D > c > E > C > e

Rh antigen frequency in Caucasian population (know this!)

• D: 85% ; absence of D: 15%

  • Highest incidence of the Rh-negative type in comparison to other populations

  • African descent--7%

• C: 70%

• E: 30%

• c: 80%

• e: 98%

importance of RhD typing

• critical in pretransfusion testing

  • principal antigen is D

  • HDFN (Rh-negative mom & Rh-positive fetus)

  • Hemolytic transfusion reaction (HTR)

• Individuals who lack RhD are deemed "Rh-negative"

  • Lack the D antigen on their RBCs

• Individuals who possess RhD are deemed "Rh-positive"

  • possess the D antigen on their RBCs

Rh pos vs Rh neg

• Rh positive individuals have RHD gene

  • D is the most important Rh antigen

• Presence of a single D antigen confers the designation of Rh-positive

• Absence of D antigen = Rh-negative

• The letter "d" is used to denote the lack of D antigen in Rh-negative individuals

Rh positive phenotype / genetics

• Rh-positive individuals may inherit one or two codominant RHD genes, which result in expression of RhD antigen

• In addition to the RHD genes(s), two RHCE genes are inherited, one from each parent

Rh negative phenotypes

• Rh-negative individuals can arise from at least three different mutations

• most often found in individuals falling into the following three different ethnic backgrounds:

  • European ethnicity → deletion of RHD gene

  • African ethnicity → RHD pseudogene (don’t produce RhD protein)

  • Asian ethnicity → alteration of RHD gene (Del phenotype)

Rh associated glycoprotein (RhAG)

• RhAG is not part of the Rh blood group system

  • It is a separate blood group system

  • The product of RHAG gene: Rh-associated glycoprotein (RhAG)

    • Located on chromosome 6

  • RhAG is a co-expressor and MUST be present for successful expression of the Rh antigens

  • Does not carry the Rh antigens

  • In the absence of the RHAG gene, multiple molecular defects in the red blood cell membrane can occur

    • May result in missing or significantly altered RhD and RhCE proteins, affecting antigen expression

where is the RHAG gene located?

chromosome 6 (!!!)

(variations of D antigen) weak D

•  rarely form anti-D

  • Position effect: c in trans to D, D is complete

  • Quantitative: D is complete, fewer number D_el

(variations of D antigen) partial D

D antigen is not complete, missing or altered epitopes, can form anti-D against the missing epitope(s)

(Rh deficiency) Rh null

• Very rare, lack LW and FY5 antigens

• Lack all Rh antigens

  • Mutation in RHAG gene (regulator-type)

  • Mutation in RHCE gene, deletion in RHD gene (amorphic type)

characteristics of Rh null syndrome

• compensated hemolytic anemia

• slight to moderate decreased hgb/hct

• reticulocytosis/stomatocytosis

• decresed haptoglobin

• increased hemoglobin F

• absence of FY5 antigen

(Rh deficiency) Rh mod

• Partial suppression of RH gene expression caused by mutations in the RHAG gene

• Characteristics similar to Rh_null phenotype

(other Rh antigens) G antigen

• Serine at position 103 on Rh polypeptides

• On most D positive and all C positive RBCs

• Combination antigen present on RBCs that have either C or D antigens

• Distinction between anti-G, anti-C, and anti-D important in pregnant Rh-negative patients

  • If anti-G is present in Rh-negative pregnant patient, then Rhogam (RhIG) administration is needed

  • If anti-C and anti-D present, then Rhogam administration is not indicated

  • Anti-G looks like an inseparable anti-D,-C

(other Rh antigens) f (ce) antigen

• When both c and e antigens are present on RBC and respective genes in cis position or on the same chromosome, the f antigen is expressed

• RHCE encodes for c, e, and f antigens

• f antigen present in majority of D-negative individuals

• Anti-f implicated in HDFN and HTRs

(other Rh antigens) Ce antigen

when C and e in cis position, Ce antigen present

(other Rh antigens) C^w antigen

• Low frequency antigen

• Antithetical to high-incidence antigen, MAR

• Found in 1-2% Caucasian population, rare in people of African descent

• Naturally occurring antibodies (anti-C^w implicated in some cases HTRs and HDFN)

(other Rh antigens) V antigen

• Rare in Caucasian population (1%): more common in people of African descent

• V antigen associated with VS antigen (not antithetical)

• IgG antibodies react best at 37 C (some reactivity at RT)

• Usually present with other antibodies such as anti-D

• Anti-V is not clinically significant

Rh antibodies (general)

• Anti-c is the second most important antibody in the Rh blood group

• Anti-E is more common

  • Can be detected as a naturally occurring antibody

• Anti-c and anti-e are produced in response to antigenic exposure

• Rh antibodies are primarily of the IgG class--able to cross the placenta

• Dosage

• Do not bind complement

landsteiner wiener (LW) blood group system

• Four alleles compose the LW blood group system

  • LW^a, LW^ab, and LW^b

• Phenotypically, there is a similarity between the Rh and LW systems

• Note the following reactions:

  • Anti-LW reacts strongly with most D-positive RBCs, weakly (sometimes not at all) with Rh-negative RBCs, and never with Rh_null cells

  • React well with cord cell regardless of Rh type

molecular genetic theories of Rh genetic control (2)

• Wiener's Agglutinogen Theory

  • One gene is responsible for defining Rh—the agglutinogen produced contains three Rh factors

  • Antibody will recognize each factor within the agglutinogen

• Fisher-Race

  • Antigens produced by closely linked genes

  • Each gene produces one antigen on the RBC surface

Wiener’s Agglutinogen Theory

Wiener postulated that a single gene produces a single product that contains separately recognizable factors

• This Rh gene produced at least three factors within an agglutinogen

  • agglutinogen may be considered the phenotypic expression of the haplotype

  • Gene = agglutinogen Rh factors

• Each factor is an antigen recognized by an antibody

• Antibodies recognize single/multiple factors or antigens

• Nomenclature (R₀, R₁, R₂, R_Z, r, r', r", r^y)

  • Rh₀ = Dce (0 = no CE)

  • hr' = Ce = 1

  • hr" = cE = 2

  • CE = z or y

Fisher-Race Theory

• Fisher and Race proposed that the Rh locus contains three distinct genes that control production of their respective antigens

  • Each gene was responsible for producing a product (or antigen) on the RBC surface

• Genes and gene product defined by same letters and order is DCE

• Nomenclature

  • More widely adopted than Wiener nomenclature

  • Used to interpret serological workups

  • Eight possible combinations possible

    • Dce, DCe, DcE, DCE, dce, dCe, dcE, dCE

Tippett’s theory (the correct one)

• There are two RH genes: RHD and RHCE

  • closely linked genes on chr 1

• These genes control expression of Rh antigens, D and C/c and E/e, respectively

  • RHD—codes for presence/absence of D antigen

  • RHCE—codes for presence/absence of C/c and E/e antigens in various combinations

    • RhCE, rhcE, Rhce, RhCe

• Both genes differ by 32 to 35 amino acids (dependent on phenotype)

• Each gene possesses 10 exons in its genetic structure

• Most Rh-negative phenotypes are the result of the complete deletion of the RHD gene

nomeclature (general; 4)

• Fisher-Race Terminology

  • Three closely linked genes within the Rh system (DCE)

• Wiener terminology

  • Individuals inherit Rh antigens as product of single gene at a single locus (Rr)

• Rosenfield terminology

  • Alpha-numeric system (1, 2, 3, 4, 5)

  • Antigens identified by letters to blood group and numbers to antigens

• International Society of Blood Transfusion (ISBT)

  • System based on genetic classification

  • Both alphanumeric and strictly numeric system used

(nomenclature) fischer-race

aka DCE terminology

• The phenotype of a given RBC is defined by the presence of D, C, e, E, and e antigenic expression

  • lack of D antigen = d

• Weakened antigen expression documented with parenthesis, e.g., (D), (C , (e)

• Haplotype is the complement of genes inherited from either parent

  • DCe--most common haplotype for Caucasian and Asian populations

  • Dce--most common haplotype for African-Americans

(nomenclature) wiener

aka Rh-Hr

• R = presence of D antigen

• r = absence of D antigen

• Italics and subscripts are used

  • Rh₀ = D

  • 0 = no C and E

  • Single prime ‘ or 1 = C

    • no (‘) or 1 = c

  • Double prime ‘‘ or 2 = E

    • no (‘‘) or 2 = e

  • z or y = presence of C and E

 

converting Weiner terminology into Fisher-Race & vice versa

• R = D / r = no D

• subscript 1 or ‘ = C

• subscript 2 or ‘‘ = E

• subscript z/y = C and E / subscript 0 = no C and E

• ex: DcE = R₂ ; r’’ = dcE

(nomenclature) rosenfield & coworkers

aka alphanumeric terminology

• In the early 1960s, Rosenfield and associates proposed a system that assigns a number to each antigen of the Rh system in order of its discovery or recognized relationship to the Rh system

• This system demonstrates the presence or absence of the antigen on the RBC

  • Rh1, Rh2, Rh3, Rh4, Rh5

  • D is Rh1, C is Rh2, E is Rh3, c is Rh4, e is Rh5

• A minus sign preceding a number designates absence of the antigen

  • e.g., -1, 2, 3, 4, 5 (D antigen is absent)

• If an antigen has not been typed, its number will not appear in the sequence

  • e.g., 1, 2, 4, 5 (E antigen has not been typed)

(nomenclature) international society of blood transfusion committee (ISBT)

• formed the Committee on Terminology for Red Cell Surface Antigens

• mandate was to establish a uniform nomenclature that is both eye- and machine-readable

• ISBT adopted a six-digit number for each authenticated antigen belonging to a blood group system

  • first three numbers represent the system and the remaining three the antigenic specificity

  • Number 004 was assigned to the Rh blood group system, and then each antigen assigned to the Rh system was given a unique number to complete the six-digit computer number

prevelance of the principal Rh haplotypes (whites)

• DCe = R₁ = 42%

• ce = r = 37%

• DcE = R₂ = 14%

• Dce = R₀ = 4%

• Ce = r’ = 2%

• DCE = R_z = <0.01

Rh phenotypes and genotypes in the US (pic)

• most prevalent: R₁r = DCe/dce

  • 35% of Caucasians ; 9% of African Americans

• least prevalent: R₀r = Dce/dce

  • 2% of Caucasians ; 23% of African Americans

Rh testing (general)

• The presence/absence of D antigen tested for all blood donors and recipients in most countries

• Rh phenotype defined by testing with anti-D, anti-C, anti-E, anti-c, and anti-e reagents

• An individual's Rh genotype determined by molecular testing (e.g. DNA analysis) or phenotyping in a family study

• Weakened expression of D

  • Rh(D) positive RBCs give readable, macroscopic reactions when tested with anti-D commercial reagent

  • Some cells may appear Rh(D) negative with some anti-D reagents and positive with others

weak D (Du) testing

• Performed to test for weak expression of the D antigen

• RBCs may react weakly (2+ or less) or not at all in direct testing with anti-D reagent

• Rh typing taken through the AHG testing phase

• In tube testing, anti-D tube incubated at 37°C to examine for agglutination

• Transfusion considerations

  • Weak D test must be performed on all Rh-negative blood donors

  • Only when D and weak D tests negative is unit given Rh(D) negative designation

  • Weak D patients – transfuse with D negative RBCs

Rh typing reagents (3)

• Monoclonal antisera

  • Made from cell culture

  • Cell line capable of growing outside a body in liquid medium

  • Derived from cancer cells or hybridoma

• High and low-protein human antisera

  • Anti-D and Rh control regents made from human plasma divided into high and low-protein formulations

• Rh control and Weak D test

  • Controls used when Rh typing test taken through AHG phase

(sources of error in Rh typing) causes of false positive reactions

• contaminated reagents

• abnormal proteins in patient/donor plasma causing rouleaux

• use of wrong antiserum

• failure to follow manufacturer’s directions

• cold agglutinins (auto/alloantibodies) in patient or donor plasma

(sources of error in Rh typing) causes of false negative reactions

• failure to add antiserum

• use of wrong antiserum

• RBC suspension too heavy

• patient/donor RBC with a variant Rh antigen that does not react w the antiserum

• failure to follow manufacturer’s directions

Rh alloantibodies

• RBC stimulated

• Must be exposed to antigen BEFORE antibody formed

• Highly immunogenic

• Individuals who have developed anti-D more prone to develop other Rh blood group system antibodies

Rh autoantibodies

• Rh specificity to a common Rh antigen or high-prevalence Rh antigen

• Weaker reaction when tested with RBCs having normal Rh antigen expression

• Controversy over transfusion

• Risk of alloimmunization

• Patients with autoantibodies increased tendency to produce alloantibodies

characteristics of rh antibodies

• are IgG and react optimally at 37 C OR after AHG testing

  • IgG1, 2, 3 and 4 subclasses of Rh antibodies have been reported

    • IgG1 and IgG3 are of the greatest clinical significance

  • RBCs coated with IgG1 and IgG3 are rapidly cleared from circulation (extravascularly) by the reticuloendothelial systems (RES)

• Are usually produced following exposure to foreign RBCs

• May demonstrate dosage

• Enhanced when testing with enzyme-treated RBCs

• Often persist for years

• Do NOT bind complement

• Can cross the placenta

transfusion considerations for Rh types

• Rh (D) positive Individuals

  • May receive D positive or D negative blood products

• Rh (D) negative Individuals

  • Should receive RBC containing products that lack D antigen to avoid immunization

Rh implicated transfusion reactions

• D antigen is the most immunogenic antigen outside the ABO system

• Clinical manifestations include the following:

  • Unexplained fever

  • Mild bilirubin elevation

  • Decreased hemoglobin and haptoglobin

  • DAT is usually positive

  • Extravascular hemolysis

• Antibody screen may demonstrate circulating antibody

• Elution studies may be helpful

characteristics of intravascular hemolysis in a transfusion reaction

ex: ABO blood group HTR

• Hemolysis occurs (inside the blood vessel)

• Anemia

• ↑ Bilirubin ; ↑ LDH

• ↓ Haptoglobin

• Plasma with free hemoglobin

• Blood in urine (hemoglobinuria) + urine hemosiderin (free heme)

• RBC Morphology: Schistocytes, Reticulocytosis

• Complement activation

• Primarily IgM antibodies

characteristics of extravascular hemolysis in a transfusion reaction

ex: Rh blood group HTR

• Hemolysis occurs via the spleen and reticuloendothelial (RES) mediated → mononuclear phagocyte system

• Anemia

• ↑ Bilirubin ; ↑ LDH

• ↓ Haptoglobin

• Normal plasma hemoglobin

• RBC Morphology: Spherocytosis, Reticulocytosis

• No complement activation

• Primarily IgG antibodies

hemolytic disease of the fetus and newborn (HDFN)

• Rh HDFN is often severe

• Rh antigens are well developed on fetal RBCs and Rh antibodies are transplacental IgG antibodies

• Clinical intervention

  • Rh-immune globulin (Rhogam) is a purified preparation of IgG anti-D

  • Administered to D-negative women during pregnancy and after delivery of a D-positive fetus

  • Rh-immune globulin is effective only in preventing RhD HDFN

Rh-immune globulin (rhogam)

• RhIG prevents immunization against D antigen ONLY and not against other blood group antigens

  • Rhogam is a solution of concentrated anti-Rh0(D)

  • Prepared from pooled human plasma from patients hyperimmunized

  • Contains predominantly IgG anti-D

dosing of rhogam (RhIG)

• Two doses (IM)

  • 50-μg

  • 300-μg: Considered a full dose protective against 15 mL of D-positive RBCs or 30 mL whole blood

• During pregnancy…

  • First 12 weeks: 50-μg IM dose indicated for abortion or miscarriage

  • After 12 weeks: a full dose (300-μg ) IM indicated for abortion or miscarriage

  • After 34 weeks: 120-μg (IV) indicated for amniocentesis or complication

  • Antepartum: 300-μg IM given at 28 weeks to non-immunized D-negative females

(HDFN assessment) rosette test

qualitative screening test demonstrating number of D-positive RBCs in a D-negative suspension with chemically modified anti-D reagent

• Detectable in 10 mL or more of fetal whole blood in maternal circulation

  • Postpartum maternal blood sample collected and incubated at 37°C with monoclonal anti-D reagent

  • Anti-D binds to the D-positive fetal RBCs

  • Maternal RBCs are washed to remove unbound antibody

  • Indicator cells (D-positive RBCs) are added, centrifuge, resuspend and read microscopically for visible agglutinates referred to as “rosettes”

  • Fetal RBCs must be D-positive, maternal RBCs must be D-negative for a valid test

(HDFN) quantitative test

• Fetomaternal Hemorrhage (FMH)

  • D-positive fetal RBCs detected in maternal circulation

  • If positive, must be quantified using the Kleihauer-Betke or flow cytometry test

• Determine Rh status of newborn

• D-positive fetal RBCs and Rh type cannot be determined on newborn (e.g., positive DAT), mother should receive full dose of RhIg

  • Exception: Previous active immunization against D antigen