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

HDFN + detection of antibodies

hemolytic disease of the fetus and newborn (HDFN)

destruction of the RBCs of a fetus or neonate by maternal antibodies that attached to corresponding antigens on baby's RBCs

• Antibodies transferred through the placenta stimulated naturally (ABO) or by previous pregnancy or transfusion during current pregnancy

• Results in anemia, accumulation of bilirubin, enlarged spleen and liver

facts of HDFN

• Pathogenesis of HDFN elucidated discovery of the Rh antigen

• Discovery of Rh-immune globulin in 1968 led to prevention of HDFN caused by anti-D

etiology of HDFN

• Sensitization of blood group antigens occurs with previous pregnancies or RBC transfusions

• Fetal RBCs possessing a paternal antigen foreign to mom can enter the maternal circulation causing feto-maternal hemorrhage (FMH)

• FMH more likely to occur at delivery once placenta separates from the uterine wall

pathophysiology of HDFN

• Associated causes of HDFN include ABO, Rh or other blood group incompatibility between mother and infant

• Four stages of pathogenesis

  1. Exposure of mother to fetal RBC antigens with subsequent production of maternal blood group antibodies

  2. Placental transfer of the maternal antibodies to the fetus w attachment to fetal RBC antigens

  3. Subsequent immune destruction of the antibody-coated fetal RBCs

  4. Clinical manifestations resulting from destruction of the "marked" RBCs

disease progression of HDFN in fetus

• RBCs are destroyed → fetus becomes anemic

  • Erythroblastosis fetalis--immature nucleated RBCs released into fetal circulation from BM

• response to anemia → RBCs cells made in liver and spleen (in utero)

  • Hepatosplenomegaly occurs

• Progressive anemia causes tissue hypoxia → increased cardiac output

  • Heart beats rapidly with eventual heart failure

• Hydrops fetalis--significant fluid buildup characterized by respiratory / circulatory distress

• At birth, affected newborns are anemic but not jaundiced

  • Kernicterus--unconjugated bilirubin crosses the BBB causing cell death and characterized by seizures, hearing loss, poor feeding, and can be fatal

conditions necessary for maternal antibody formation in HDFN (4)

1. the mother must lack the antigen present on the fetal RBCs

2. the fetal antigen must be well developed in utero

3. the mother is exposed to the fetal antigen

4. the mother produces and IgG antibody to the antigen, capable of crossing the placenta

HDFN antigens

• Antigens localized to RBC membrane are more antigenic

• Early maturation of antigens plays a role in HDFN severity

  • e.g. Kell antigen develops early in fetal development

• Density of the antigen plays a role as well

  • Homozygous vs heterozygous expression

HDFN antibodies

• IgG crosses from mother's circulation → fetal capillaries in placenta

• Neonatal Fc receptor (FcRn) transports IgG from mother to fetus and maintains homeostasis of IgG in circulation

• Immunoglobulin transferred to fetus early in 2nd trimester

• ABO-incompatible blood group of fetus reduces incidence of maternal formation of anti-D

  • Group O, Rh neg mothers who carry group A or B, Rh-pos fetuses

  • ABO incompatible RhD-pos fetal RBCs destroyed and cleared in the mother's circulation before the RhD antigen can be recognized by maternal immune system

ABO HDFN

• Before 1968, 95% of HDFN was due to anti-D

• Now, most common HDFN is caused by ABO antibody (1:5 pregnancies, mild)

• Group O mom with potent high-titered IgG anti-A,B

• Infants group A (Caucasian); group B (African-American)

  • Spherocytes

  • Bilirubin peaks later (1-3 days)

  • Mild compared to Rh HDFN

  • DAT positive

Rh HDFN

• Approx 15% of the population is Rh negative (no D antigen)

• Rh-negative mothers carrying Rh-positive fetuses run the risk of producing anti-D

  • Feto-maternal bleeding can occur with any pregnancy

  • Maternal and fetal blood may mix at delivery

• In the mid-1960s, it was discovered that if non-sensitized Rh-negative persons received intramuscular doses of Rh-immune globulin (RhIG), their immune systems failed to produce anti-D antibodies (even after exposure to the D antigen)

• Rh-negative pregnant women are now immunized during and immediately after delivery with RhIG

rhogam treatment of Rh HDFN (general)

must determine quantity of Rh-immune globulin to administer

• The feto-maternal bleeding usually less than 30 ml of whole blood

• One regular-dose vial of RhIG contains 300 ug of RhIG → sufficient to protect against 30 mL of whole blood or 15 mL of packed RBCs

  • One unit of RhIG is generally sufficient during pregnancy, 28 weeks gestation

  • The second dose is administered within 72 hours of delivery

• In incidences of increased FMH bleeding, the quantity of RhIG units must be calculated to prevent formation of antibodies

kleihauer-betke acid-elution test

• Quantitates fetal cells in the maternal circulation by using a blood smear made from blood drawn from the mother

• Smear is placed in an acid buffer

  • Hemoglobin in adult RBCs (Hgb A) leaches out of the cells into the buffer

  • Fetal Hgb (Hgb F) is resistant to the acid and will stay within the RBC

• Smear is washed, stained with Wright stain, and examined microscopically under oil immersion (1000x)

• Adult RBCs will look "ghostlike" because only the cell membrane remains, and the fetal cells will stain pink

kleihauer-betke acid-elution test calculation

determines the % of fetal cells

• The number of fetal cells counted per 2000 maternal cells is quantitated similar to a reticulocyte count:

  • % fetal cells = (# of fetal cells counted / 2000 maternal cells) x 100

rhogam dose calculation

• Units or vials of RhIG to be administered are calculated by the following formula:

  • vials of RhIG = (% fetal cells x 50) / 30

  • % fetal cells = number of fetal cells per 2000 maternal cells

  • 50 = factor to account for average maternal blood volume

  • 30 = quantity of fetal-maternal bleed

• Result is rounded to the nearest whole number

• Additional unit is added for safety

rhogam prophylaxis

• Antepartum prophylaxis

  • 300-ug dose of RhIG between 28 and 30 weeks in pregnant Rh-negative women who have not made anti-D

• Postpartum prophylaxis

  • 300-ug dose of RhIG within 72 hours to Rh-negative women who deliver Rh-positive infant

  • Have not produced anti-D

non-ABO HDFN prenatal evaluation

• Maternal history involves review of any previous infant affected by HDFN and infants who required transfusion

• Serological studies include ABO, Rh, and antibody screen testing (if required)

• Paternal phenotype

  • Likelihood fetus carries antigen to which mother has formed antibody

• Typing of fetus

  • Obtaining a sample of RBCs from fetus can be risk to fetus and/or mother

  • Amniocentesis procedure of choice to obtain amniotic cells containing fetal DNA

  • Fetal DNA can be isolated from maternal plasma

HDFN monitoring using antibody titration

• Pregnant women w history of anti-D or any other IgG antibody will have baseline titer performed during the first trimester

  • Recommended to freeze aliquots for future titers every 4-6 weeks during pregnancy

• 1:2 dilution series of patient serum w saline, 37 C, IAT, anti-IgG reagent

• An increase in new titer by 2 dilutions or higher compared w original sample is considered significant

measurements of severity in HDFN

• Amniotic fluid analysis

  • Level of bilirubin pigment in amniotic fluid correlates with hemolysis of fetal RBCs in utero

• Doppler flow studies

  • Ultrasound noninvasive method of monitoring anemia in fetus

• Fetal blood sampling

  • Most accurate means of determining severity of fetal hemolytic anemia and need for treatment of fetus

antepartum treatment of HDFN

• Plasma exchange can be safety performed in pregnant women

  • Goal of removing maternal alloantibody from circulation

• Intrauterine transfusion (IUT) or direct intravascular fetal transfusion may be performed as early as 20th week of gestation

• Selection of RBCs important

  • Group O Rh-negative lacking the offending antigen

  • Less than 5-7 days old (or washed)

  • Leukocyte-reduced (LR) w CMV-safe, irradiated, hemoglobin S negative and compatible with mother's plasma

intrauterine transfusions (IUT)

• Blood must be compatible w maternal antibodies capable of crossing the placenta

• Test for maternal antibodies with donor antigens

• Ensure no feto-maternal ABO or Rh incompatibility

• Crossmatch testing is performed using the mother's sample

serological testing of newborn (cord blood)

• Direct antiglobulin test (DAT)

  • If mother has significant antibody, DAT performed on newborn's sample with anti-IgG reagent

  • Rh-positive baby born to mother who received RhIG may have positive DAT due to passive transfer of anti-D

  • If neonate has positive DAT and mother has negative antibody screen, antibody to low-incidence antigen may be suspected

• ABO and Rh determination

  • Only typing of RBCs is required

treatment of HDFN (after baby is born)

• Phototherapy

  • All infants monitored for development of jaundice

  • Phototherapy or light-based treatment alone

• Small volume transfusion

  • Depends upon degree and rate of hemolysis and resultant increase in bilirubin level

• Exchange transfusion

  • Replacing one or more volumes of infant's blood with compatible RBCs and plasma

  • Removes antibody-coated RBCs of infant

  • Removes maternal antibody present in infant's circulation

  • Corrects anemia without causing volume overload

neonatal transufsions

blood for an exchange or regular transfusion of a neonate should be compatible with any maternal antibodies that may have entered the infants' circulation and optimally react at 37 C or AHG

• Testing considerations

  • ABO and Rh testing required

    • ABO group of neonate determined only by typing RBC w commercial anti-A and anti-B

  • Antibody detecting testing required

  • Indications for red blood cell transfusion

    • Any baby showing signs or symptoms of anemia

RBC components for neonatal transfusion

• RBC transfusions in infants less than 4 mo of age

  • Compatibility testing

    • ABO and Rh type of RBCs from cord/heel stick

    • Antibody screening on serum or plasma of mother or on eluate prepared from infant's RBCs

    • Crossmatch compatible w serum or plasma of mother

• Stored RBCs for small volume infant transfusions, group O Rh-negative

• Leukocyte-reduced, preferably by pre-storage filtration, fresh (5-7 days)

• Irradiated, CMV-safe, hemoglobin S negative

  • Irradiated cellular blood components indicated for infant at risk of transfusion-associated graft vs host disease (TA-GVHD)

  • Cytomegalovirus (CMV) infection can be devastating disease in fetus or newborn

prevention of Rh HDFN

use of Rh-immune globulin (RhIG)

• Decreased incidence of HDFN resulting from production of anti-D by Rh-negative mother

• Sterile solution containing IgG anti-D

ABO vs Rh HDFN

• ABO

  • first pregnancy: rare

  • canNOT predict by titer

  • main ABY: anti-A,B

  • billirubin normal + no anemia at birth

  • IUT: none

  • exchange transfusion rare; phototherapy needed

• Rh

  • first pregnancy: rare

  • CAN predict by titer

  • main ABY: anti-IgG

  • bilirubin elevated + anemia at birth

  • IUT: sometimes

  • exchange transfusions uncommon; phototherapy needed

immune alloantibodies

produced in response to RBC stimulation via transfusion, transplantation, or pregnancy

naturally occurring antibodies

exposure to environment sources that have similar structures to RBC antigens

passively acquired antibodies

via plasma-containing blood components or derivatives (e.g. IVIG)

autoantibodies

directed against one's own RBCs and all the RBCs tested

clinically significant antibodies

IgG, 37 C/AHG (IAT), reduce survival of RBCs

antibody screen (ABS)

detect clinically significant antibodies in the blood donor and the intended recipient

• included in standard prenatal testing to evaluate the risk of HDFN in the fetus and to assess the mother's candidacy for Rh-immune globulin (RhIG) prophylaxis

• overall purpose: assess for unexpected alloantibodies or (autoantibodies) that may be clinically significant

(antibody screen methods) tube method

• 2 drops plasma + 1 drop of Group O reagent screening cells

• Use of enhancement media

• Testing phases

  • IS, 37 C, and AHG

• Confirm all negative reacting tubes with Coomb's control cells (check cells)

• Agglutination visualized as "clumps of cells"

  • ABO typing/grouping ; Rh(D) typing

  • Antibody screen and identification

  • Crossmatching

  • Antigen typing ; DAT

• Enhancement media is often used: LISS, PeG, and albumin

(antibody screen) tube testing methodology

• Traditional/gold standard of blood bank testing

• IgM (immediate spin or cold reactive antibodies; see first pic)

• IgG (indirect antiglobulin test/IAT; second pic)

antigen profiles (reagent rbc panel)

• Antigen profiles (antigrams)

  • Grids that indicate antigen make up of commercial RBCs used for antibody detection or identification

  • Uses group O reagent RBCs (phenotyped)

  • A well-designed panel will identify most commonly encountered antibodies

• Antibody screen—2 or 3 cell panels

• Antibody identification—can be 10, 16, or 20 cell panels

anatomy of antigram

• "+" sign refers to the presence of the antigen

• "0" sign refers to the absence of the antigen

(antibody screenmethods) column agglutination technology (CAT) gel

• Agglutination visualized by capture of RBCs within gel column

• Used for same routine tests as tube testing

• involves microtubules filled with a dextran acrylamide gel

• The screen cells used for this technique meet the same criteria as for the tube test BUT are suspended in LISS to a concentration of 0.8%

• Large agglutinates remain near or on top of the gel matrix

• Smaller agglutinates pass through the gel matrix

• Unagglutinated cells pass through the gel matrix to form a button at the bottom of the microtubules

(antibody screen methods) solid phase red cell adherence (SPRCA)

• Used for antibody screening and antibody identification

• Relies on antigen-coated wells in solid phase microplates

• Can be manually performed or on an automated analyzer (Immucor Neo, Immucor Echo)

• Testing Process:

  • Screening cells bound to surface of polystyrene microplates

  • Pt plasma and LISS added to the wells

  • RBC antigens capture IgG ABYs during incubation step

  • Microplates washed to remove unbound ABYs

  • Indicator cells (or anti-IgG coated red cells) added to wells

  • Microplates centrifuged to bring indicator cells in contact with bound antibody (if present)

pros/cons of tube method for antibody screen

• detects IgG & IgM; needs 100 uL of plasma/serum

• uses 3-5% suspension of RBCs

• advantages:

  • gold standard, easily manipulated, differentiates IgM from IgG

• disadvantages:

  • no automation available, human techniques and subjectivity, unstable rxns

pros/cons of gel method for antibody screen

• detects IgG & IgM; needs 25 uL of plasma/serum

• uses 0.8% suspension of RBCs

• advantages:

  • small sample volume, very sensitive, stable rxns, automation available

• disadvantages:

  • no wash step, IgM interference in IgG tests

pros/cons of solid phase method for antibody screen

• detects IgG only; needs 50 uL of plasma/serum

• uses 3% suspension of RBCs

• advantages:

  • automated, sensitive, designed for IgG, stable reactions

• disadvantages:

  • very sensitive to autoantibodies, must prepare own monlayers or buy commerically prepared 

autocontrol (AC)

• An autologous control consists of testing the patient's serum (or plasma) against their own RBCs by the same method used to test ABID panel cells

  • Tested whenever an ABID panel is performed

  • 2 drops plasma or serum + 1 drop RBC suspension

• Autoantibodies, when present, may complicate the detection of clinically significant alloantibodies

  • Hence the setup of testing an autocontrol

questions to ask to interpret antibody screens

• In what phase(s) did the reaction(s) occur?

• Is the autologous control negative or positive?

• Did more than one screen cell react?

  • If so, did they react at the same strength/phase(s)?

• Is hemolysis or mixed-field agglutination present?

  • Hemolysis? Anti-Lea, anti-Leb, anti-PP1Pk, anti-Vel

  • MF? Lutheran, anti-Sda

• Are the cells truly agglutinated or is rouleaux present?

  • Does not interfere with AHG phase in tube or solid-phase testing due to patient’s serum being washed away prior to addition of AHG

  • Saline wash

limitations of an antibody screen

• Will not detect ALL antibodies!!

  • If a certain Ag (low prevalence) is not represented on screening cells, the Ab (if present) will not be detected

  • Antibodies that show dosage

  • Antibodies with low titers

    • Screen may look negative; antigen positive units given => result in delayed hemolytic transfusion reaction days or weeks later

antibody screen results + possible interpretations:

• 2+ only at AHG phase with SCII

1. single alloantibody

2. two alloantibodies, antigens only present on cell II

3. probably IgG alloantibody 

antibody screen results + possible interpretations:

• 1+ at 37 C with SCI

• 3+ at AHG with SCI

• 1+ at AHG with SCII

1. multiple alloantibodies 

2. single alloantibody (dosage)

3. probably IgG alloantibody 

antibody screen results + possible interpretations:

• 1+ at IS with SCI

• 2+ at IS with SCII

1. single or multiple antibodies

2. probably IgM alloantibody

antibody screen results + possible interpretations:

• 2+ at IS w SCI

• 1+ at AHG w SCI

• 3+ at IS; 1+ at 37 C; 2+ at AHT w SCII

1. multiple alloanitbodies, warm and cold

2. potent cold alloantibody binding complement in AHG

antibody screen results + possible interpretations

• 1+ at AHG w SCI and SCII

1. single warm alloantibody, antigen present on both cells

2. antibody to high-prevalence antigen

3. complement binding by a cold alloantibody not detected at IS

antibody screen results + possible interpretations

• 3+ at AHG for SCI, SCII, and with autocontrol

1. warm alloantibodies

2. transfusion reaction

3. probably IgG alloantibody

4. warm autoantibody 

what is done after a positive antibody screen?

reflex to ABID panel for identification of antibodies using IAT procedure

• Patient serum or plasma mixed with Group O Screening Cells (RBCs) with known antigens

• Test phases and enhancement reagents

  • Tube, gel, and solid phase methods are approved for ABID testing

  • Gel and solid phase testing are very sensitive techniques

  • Tube testing has more variation in technique but still helpful in identifying antibodies

purpose of ABID

• Performed to determine the specificity of the antibody/antibodies

  • Single alloantibody may be simple to ID

  • Multiple alloantibodies or autoantibodies may be complex and require a great deal of time and expertise

• Important when selecting donor units for transfusion

• Monitoring potential HDFN cases

how is an ABID panel done?

• Testing of plasma against panel of 11 to 20 group O RBCs with KNOWN phenotypes 

  • Focused on DETECTION of unexpected antibodies

• Each panel should have antigram with various antigen expression

  • Shows antigenic makeup of each cell in panel

  • Should include cells with homozygous expression

what patient history is relevant to know for an ABID panel?

• Has patient ever been transfused or pregnant?

• If patient has been transfused, did any transfusions occur in the last 3 months?

• What medication is the patient currently taking?

• Are there any other medications patient has taken in the last 3 months?

ABID panel selection

• The purpose of the ABID panel is to identify unexpected clinically significant antibodies

• Antibody screen and ABID panel cells are prepared from group O donors

  • Cells do not contain A or B antigens on RBC surface

  • Eliminates chance of interference from ABO antibodies in serum or plasma

• Factors influencing sensitivity

  • Serum to cell ratio

  • Temperature and phase of reactivity

  • Length of incubation and pH

difference between antibody screen & antibody identification

• Screening uses two to three different donor cells

• Antibody identification uses cells from a larger number of donors (10 to 20 donors)

(ABID panel evaluation) questions to ask for inital interpretation of panel

• Where did the reactions occur?

  • Is the autologous control negative or positive?

• In what phase(s) and at what strength(s) did the positive reaction(s) occur?

  • AHG phase positive? = IgG antibody

  • All same strength? = single antibody

• Did more than one panel cell react?

  • If so, did they react at the same strength/phase(s)?

    • In tube method, IS phase positive only = COLD IgM; not clinically significant!

    • If positive in all three phases; may have two antibodies (cold and warm) or a cold with wide thermal range – need to figure it out

(ABID panel evaluation) questions to consider after ruling out antibodies

• Did positive antibody reactions match a pattern of any antigens that were not ruled out?

  • If single alloantibody present, the pattern of reactivity may match exactly

  • If not an exact match, consider:

    • Dosage

    • Multiple alloantibodies

    • Cold reactive autoantibodies

    • Antibodies directed against high frequency or low frequency antigens

• Were all commonly encountered RBC antibodies excluded?

  • Low-prevalence antigens

• Was there sufficient evidence to prove the suspected antibody(ies)?

  • 3 and 3 rule

• Does the patient lack the antigen(s) corresponding to the antibody(ies)?

optimal phases of reactivity for various antibodies

• IS (RT): Le^a, Le^b; M, N; Lu^a; P₁

• 37 C: potent cold (IgM) antibodies esp those causing hemolysis; high titer warm antibodies (e.g., D, E, K)

• AHG/IAT: Rh antibodies, Kell, Duffy, Kidd, S,s; Lu^b

exclusion/”rule out” general rules

• only screening/ID panel cells that gave NEGATIVE reactions with patient plasma in ALL phases should be used to rule out

• Dosage—rule out using panel cells with HOMOZYGOUS (double dose of antigen) present

  • e.g., Rh (except D), Duffy, Kidd, MNS, and Lutheran

  • Antibodies that react more strongly with cells having homozygous antigen expression are said to “show dosage”

• After each negatively reacting cell has been evaluated, the remaining antigens should be examined to see if the pattern of reactivity matches a pattern of antigen-positive cells (inclusion technique)

exclusion/”rule out” exceptions

• There are exceptions to rule advocating use of only homozygous cells for exclusion of antibody

  • e.g., D and P1 antigens do not have antithetical alleles

• Careful exception: you may rule out potential anti-K with heterozygous Kk ag

• Antibodies to low frequency antigens (less than 2-5% of population) are uncommon

  • e.g., Anti-C^w, Anti-V, anti-VS, anti- Lu^a, anti-Js^a, anti-Kp^a

  • Most of the time you do not need to actually rule them out

exclusion/”rule out” rules for commonly encountered antibodies

• may need to use additional panel cells for more evidence to “rule in” or “rule out”

  • Select panel cells have homozygous antigens expression corresponding to your suspected antibody

  • If you have multiple antibodies, the select panel cells will also need to be negative for the antigens for the other antibodies in question

statistical measures for ABID

• Probability or p value: statistical measure that predicts likelihood of an outcome

• To be certain 95% of the time:

  • Rule of Three: antibody must react with three (3) antigen-positive cells and must not react with three (3) antigen-negative cells to be statistically proven

(techniques for resolving ABID) selected cell panels

• The cells selected for testing should have minimal overlap in the antigens they possess

• Selected cell panels are also useful when a patient has a known antibody, and the blood bank is attempting to determine if additional antibodies are present

(techniques for resolving ABID) antigen phenotyping

• Determines phenotype of tested RBCs

  • Helps prove antibody present (Does the patient RBC lack the antigen corresponding to the alloantibody in the plasma?)

  • Rules out antibodies not present (Patients will not make an alloantibody against an antigen that they possess!)

  • Special consideration: if transfused within the last 3 months or has a positive DAT

• Test specific known antisera against patient or donor RBCs

summary of the steps of antibody identification

• Positive antibody screen

• Next, test serum/plasma against a panel of reagent red cells

  • Tube--RT, 37 C and AHG phases/CCC

  • Gel and solid phase = 37 C/AHG

• Perform "ruling out" technique

  • Test selected cell panel

• Antigen phenotyping

  • Patient red cells

  • Donor red cells: should be antigen negative for antibody determined!