Blood bank Exam 3
Kleihauer-Betke Acid Elution
(Fetal RBCs/2000) * 100 = % Multiply x 50 (represents 5000 mL = blood volume of mother) Divide by 30, volume of whole blood covered by 1 vial of RhIg, to determine number of vials required. Round up or down and ADD 1 VIAL for safety.
What percent of normal and hospitalized blood donors have positive DAT's with no evidence of hemolytic anemia
0.1% of normal blood donors up to 15% of hospitalized patients
Discuss the 3 mechanisms of drug induced hemolytic anemia
1)The drug induces an autoimmune response but the antibody is directed against the RBC membrane 2)The drug binds to the RBC membrane and the antibody formed is directed against the drug 3)The antibody forms to part of the RBC membrane and to the drug
Describe the criteria for selection of units for an intrauterine or exchange transfusion
< 7 days from donation negative for mothers antibodies Irradiated
State the" length of time since the last transfusion" that is used as a cut off to determine if patient's RBCs can be accurately phenotyped
> 3 months because the RBC life span is ~120 days
Describe the Kleihauer Betke test at a molecular level
A thin smear of blood is exposed to acid buffer and the adult hemoglobin is eluted out and the smear is stained with hematoxylin and erythrosin B Adult cells will appear washed out due to the lack of hemoglobin where as fetal cells will be pink and retractile
Compare and contrast ABO versus Rh HDFN in terms of: a. Pathogenesis b. Incidence c. Blood types of baby and mother d. Severity of disease e. Prevention and treatment f. Laboratory findings: DAT and bilirubin
ABO a: incompatible Blood type b: first pregnancy c: baby=A or B . mother=O d: mild e: none f: DAT- Bilirubin is normal Rh a: IgG immunization b: subsequent pregnancies c: Baby Rh + Mother Rh- d: mild-severe e: RHIG + type and screen f: DAT + Bilirubin is elevated
Donath-Landsteiner Test
All tubes contain patient's serum and reagent O RBCs that have the P antigen Tubes 1 have 10 mL of patient serum Tubes 2 have 5 mL of patient serum and 5 mL of fresh normal serum Tubes 3 have 10 mL of fresh normal serum(control) Fresh normal serum is included as a source of complement, PCH patients may have low levels of serum complement A tubes are incubated biphasically ; first at 0-4°C for 30 min., then at 37°C for 1 hour B tubes - 0-4°C for 90 min C tubes- 37°C for 90 minutes
List laboratory findings on infants with HDFN
Anemia Hyperbilirubinemia 16-40% reticulocytes Increased nucleated RBCs Thrombocytopenia Leukopenia Hypoalbuminemia Smear: polychromasia No spherocytes (in non-ABO HDFN) asphyxia pulmonary hypertension jaundice kernicterus hyperglycemia
Autoantibodies
Antibodies that are directed against the individual's own RBCs are termed autoantibodies or autoagglutinins Identification of an autoantibody may explain decreased RBC survival in vivo. If a patient's RBCs are coated with autoantibody, the patient may present with: Serologic ABO discrepancy A positive Rh control A positive DAT A positive autocontrol on the antibody screen All cells on antibody screen and panel including the autocontrol in some patients with hemolytic anemia, autoantibodies cannot be demonstrated by routine techniques.
PCH - Serum/Plasma Testing
Autoantibody described as "biphasic hemolysin". Binds to RBCs at low temperatures. Binds complement. As cells warm up hemolysis occurs. Basis of Donath-Landsteiner test. DAT Autoantibody is IgG which acts as cold agglutinin. IgG binds to RBCs in colder parts of body. Causes complement to be bound irreversibly. IgG elutes off of RBCs in warmer parts of the body. ONLY COMPLEMENT is detected in DAT. Donath-Landsteiner Usually add fresh complement Antibody binds at cold temperatures Test is warmed to 37C. As warming occurs complement is activated and lysis of RBCs occurs.
Cold Autoadsorption Test
CANNOT be performed on recently transfused patients. Collect EDTA sample, keep warm. Separate patient plasma from patient RBCs. Wash patient RBCs with warm saline. Add aliquot of patient plasma to washed RBCs incubate at 4°C for 1 hour. Harvest serum/plasma and test against screen cells (IAT). Negative - no alloantibody. Positive and negative - alloantibody present, run panel. All positive, unsuccessful, repeat with 1X adsorbed sample
explain why Anti-G is significant in pregnancy
Can cause HDFN and HTR
Explain the techniques used to investigate serologic findings, resolve discrepancies, and detect underlying clinically significant alloantibodies in the presence of cold autoantibodies
Cold auto absorption and Pre-warming techniques
Explain procedures used to investigate serologic findings and detect underlying clinically significant alloantibodies in the presence of warm autoantibodies
Differential absorption Add patient serum to phenotyped reagent RBC's Incubate to absorb out allo/autoantibodies Remove serum Test each aliquot against an antibody panel
DAT
Direct antiglobulin test detects the in vivo coating of an individual's RBCs with IgG or complement C3
drug-induced
Drug-induced autoagglutinins are present in about 12% of the reported cases of AIHA
Explain the use of DTT in determining questionable clinical significance of an antibody such as anti-M
Dthiothreitol (DTT)- sulfhydryl compound used to disrupt the disulfide bonds of immunoglobluin M (IgM) Can be used to differentiate between an IgG and IgM antibody such as anti-M
Mechanisms for drug-induced hemolysis with regards to Ig Class, DAT results, serum and eluate results, frequency of hemolysis and give examples of medications causing Type 1
For 1 the DAT will be positive for IgG, positive reactions with all untreated RBC's the drug induces production of autoantibodies that recognize RBC antigens Serological findings are indistinguishable from WAIHA
Mechanisms for drug-induced hemolysis with regards to Ig Class, DAT results, serum and eluate results, frequency of hemolysis and give examples of medications causing Type 2
For 2 Strongly positive DAT due to IgG coating RBC's Antibody screen will be negtive High titer IgG antibodies present that only react with drug coated RBC's invitro Elute reactive with drug coated RBC's only and not reactive with reagent RBC's Hemolysis is sub-accute onset and can be life threatening Cephalosporins cause this type
Mechanisms for drug-induced hemolysis with regards to Ig Class, DAT results, serum and eluate results, frequency of hemolysis and give examples of medications causing Type 3
For 3 Positive reactions when antibody in plasma, on RBC membrane, and the drug are all present in the test system complement activation causes clinically affected patients to present with acute intravascular hemolysis Half of patients have renal failure DAT only reactive to complement present on RBCs, usually positive polly-specific AHG and anti-C3d
List the type of "whole blood" needed for an exchange transfusion
Group O packed RBCs are combined with AB plasma to create
Autoadsorption/ZZAP
If patient NOT recently transfused perform WARM autoadsorption. If RBCs heavily coated may need to pretreat with ZZAP to remove Ig. ZZAP is a combination of ficin and DTT Incubate patient serum/plasma with patient RBCs. Antibody directed against patient RBCs Incubate at 37C for 60 minutes Will attach and be removed from serum/plasma Separate (harvest) adsorbed serum/plasma Test adsorbed serum/plasma against screen cells. Negative - autoantibody only Negative AND positive reactions - alloantibody present perform panel on adsorbed serum and identify. All positive - procedure unsuccessful, repeat entire process with 1X adsorbed serum.
Paroxysmal Cold Hemoglobinuria (PCH)
Least common type of AIHA, with an incidence between 1% to 2 % Most often seen in children in association with viral illnesses Classic antibody produced: the Donath-Landsteiner antibody -an autoantibody with anti-P specificity Develop cross-reactivity to the P antigen(autoantibody to the p antigen) Caused by a biphasic hemolysin which induces hemolysis after exposure to cold. Results in hemoglobinuria and hemoglobinemia.
Rh specificity
May be directed against Rh antigen complex. Some times has a little e affinity (autoanti-e) Negative when tested against R2R2 donor cells
What should you do if a cold benign agglutinins interferes with routine serum and cell testing preformed at RT that affects the ABO?
May need to incubate sample at 37°C or do warm washes for forward typing May need to do a cold autoabsorption for reverse type
What is the treatment for most with CHD?
Most patients require no treatment and are instructed to avoid the cold, keep warm, or move to a milder climate
State the possible impact of a positive antibody screen on the fetus
Need to give antigen negative units that are AHG crossmatch compatible Usually crossmatched against mother's serum
kernicterus
Occurs in infants around 2-8 days of life With moderate to severe hemolysis, the unconjugated, or indirect, bilirubin can reach levels toxic to the infant's brain (generally, more than 18 to 20 mg/dL); if left untreated, can cause kernicterus
Describe the FMH (fetomaternal hemorrhage) fetal screening test at a molecular level (Rosette test)
One drop of the maternal RBCs incubated with anti-D, which coats the antigens on any fetal RBCs present Wash Add d+ indicator cells View microscopically Will see rosettes shaped clumps where indictor cells bound to the second Fab arm of the D+ antibodies coating the fetal RBCs Can detect a FMH of approx. 10mlsv
List the ways in which a pregnant woman may become immunized to antigens from fetal RBCs and describe the method of immunization
Other than birth can be due to: Amniocentesis Miscarriage Abortion Chorionic villus sampling Cordocentesis Blunt trauma to the abdomen Rupture of an ectopic pregnancy
Differentiate between a passive anti-D due to RhIg versus an immune anti-D
Passive anti-D shows a decreasing titer as the RhIg is removed from the blood An immune anti-D will show before the administration of RHIg and will increase as the pregnancy continues
Explain why paternal and fetal testing may be done
Paternal testing would be done for Rh or ABO compatibility Fetal if the mother has a known sever antibody
Outline the testing protocol for a neonatal transfusion workup.
Peripheral blood only forward typing Use gel to minimize required sample size Preform DAT
Considerations for transfusion therapy for patients with CHD?
Plasma exchange, blood warmer, corticosteroids
State the blood bank tests done on a pregnant woman during the prenatal, antenatal, and postpartum period
Prenatal: ABO, Rh, and antibody screen Antenatal: none unless hemorrhage is present Postpartum: Rh and FMH
State the definition and etiology of hemolytic disease of the newborn
RBC's of the fetus are destroyed by antibodies produced by the mothers immune system
Discuss treatment for Drug-Induced Immune Hemolytic Anemia (DIIAHA)
Stop the drug therapy
Explain the ameliorating effect of ABO incompatibility in Rh incompatible mother/child pairs
The ABO incompatibility can protect against Rh immunization If the mother is O and the baby A or B, the naturally occurring maternal antibodies (Anti-A or Anti-B) to fetal RBCs in mother's circulation destroy the fetal RBCs before she can become immunized to them and make any Anti-D
Explain the mechanism of RhIg protection in an Rh negative woman
The IgG anti-D coats D pos RBCs causing their destruction before being recognized
State the host factors that can affect antibody production
The ability of individuals to produce antibodies in response to antigenic exposure varies, depending on complex genetic factors. Of the four subclasses of IgG antibody, IgG1 and IgG3 are more efficient in RBC hemolysis than are IgG2 and IgG4. The subclass(es) the mother produces can affect the severity of the hemolytic disease
Describe how an Rh positive infant can falsely type as Rh negative at immediate spin phase
The baby's RBCs can be so coated in the mothers D antibodies that there are no receptor sites available for the Reagent Anti-D to bind causing a false negative When AHG is added the rxn will be strongly positive
Describe the fetal testing that is done to monitor the fetus in the event of the discovery of a clinically significant maternal antibody. Include titer, CVA-PSV, amniocentesis, cordocentesis, and intrauterine transfusion
Titers are done to monitor antibody levels CVA-PSV is done to check for anemia amniocentesis is done to check for bilirubin is necessary Cordocentesis is done if CVA-PSV is critical Intrauterine transfusion is done if CVA-PSV indicates anemia, if hydrops is noted fetal hemoglobin <10g/dL
Mathematically explain titer and dilution
Titration/titer is mathematically calculated based on the reactivity at varying dilutions
What should you do if a cold benign agglutinins interferes with routine serum and cell testing preformed at RT that affects Direct Antiglobulin Test (DAT)
Use an EDTA tube and it will usually correct the problem
How do you avoid reactivity with cold autoantibodies?
Use anti-IgG AHG instead of polyspecific ~Most cold antibodies react with polyspecific AHG because they fix complement Skipping the IS phase avoids the attachment of cold autoantibodies to the red cells Use 22% BSA instead of LISS
Describe the studies done on cord blood and their implications
Used to ABO/ Rh type the baby If positive mother may need to receive Rogam can also determine if the baby has HDN
When is a patient a candidate for RHIG
Used to protect D negative individuals exposed to D positive rbcs. Prenatally to D neg moms Postnatally when D neg gives birth to D pos After transfusion of D neg with blood products with D pos RBCs
Describe the clinical and laboratory findings in WAIHA, including indicators of RBC hemolysis, difficulties in serologic testing, selection of blood for transfusion, and possible treatments
WAIHA agglutinate everything Extravascular hemolysis Will interfere with the identification of alloantibodies May interfere with ABO and RH forward typing Requires least incompatible blood for transfusion Phenotyping usually required
Elution
When DAT is positive Elution techniques "free" antibodies from the sensitized red cells so that the antibodies can be identified
Reticulocyte harvest
Works to separate out reticulocytes in order to phenotype the patient accurately and not test any of the transfusion donor's cells
What should you do if a cold benign agglutinins interferes with routine serum and cell testing preformed at RT that affects Antibody detection and identification
You can use the prewarm technique or cold autoabsorption
What should you do if a cold benign agglutinins interferes with routine serum and cell testing preformed at RT affects that Compatibility testing
You use the prewarm technique or cold autoabsorption
Describe RBC morphology in HDFN
anicsocytosis increased nucleated RBC's pollychromasia
When testing is performed at 4°C, what is the most commonly encountered autoantibody?
benign cold agglutinin that may be found in the serum of most normal, healthy individuals The typical cold agglutinin has a relatively low titer (<64 at 4°C)
Define "least incompatible" with respect to transfusion
blood that has the weakest reaction in AHG during crossmatch. 1+ rather than a 3/4+
jaundice
build up of bilirubin due to hemolysis or liver damage characterized by yellowish skin and whites of the eyes
Describe the anti-f
can cause HDFN and HTR Not commercially available
hydrops fetalis
condition caused by RBC destruction outside of the bone marrow resulting in spleen and liver
erythroblastosis fetalis
condition where fetal erythroblasts are released into circulation
List the goals of an exchange transfusion
decrease accumulated bilirubin reduce amount of unbound maternal antibody remove antibody-coated cells replace infants cells with cells compatible to the maternal antibody
Allogenic Adsorption
done on recently transfused patient differential allogenic absorption select 3 donor RBC that lack common RBC antigens and are complimentary
Symptoms of PCH
episodes occur upon exposure to cold and are usually acute: Fever Shaking chills Malaise Abdominal cramps Back pain Signs of intravascular hemolysis along with hemoglobinemia and hemoglobinuria Can lead to splenomegaly, hyperbilirubinemia and renal insufficiency
Describe the f antigen
expressed when both little c and little e are present on the same haplo type is present in the vast majority of D-negative ("Rh-negative") individuals
Explain hydrops fetalis and why the liver and spleen are enlarged
extra-medullary hematapoesis that causes hepato and splenomegaly Severe anemia and hypoproteinemia lead to the development of high-output cardiac failure with generalized edema, effusions, and ascites
indirect bilirubin
free unbound bilirubin in circulation
Contrast metabolism of hemoglobin with regards to bilirubin between a fetus and a newborn infant
in the fetus the mother's liver conjugates the bilirubin and the newborn's liver isn't mature enough to conjugate it
Describe the G
is present on most D-positive and all C-positive people
State the definition, characteristics and most common causes of HDFN
maternal antibodies cover the baby's RBCs and that causes hemolysis Intra-placental hemorrhage Blunt trauma to the mothers stomach
warm reactive
mostly due to anti-IgG but can also present with anti-complement optimal temperature of reactivity: (30°C to 37°C). About 70% of the reported cases of AIHA react best at warm temperatures
cold reactive
mostly due to anti-compliment optimal temperature of reactivity:(4°C to 30°C) ~autoagglutinins account for about 18% of cases frequently encountered in serologic testing Most are not clinically significant, but occasionally they are clinically significant and cause immune hemolytic anemia Cold autoantibodies are IgM and can activate complement
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD)
often associated with an infectious disease, such as Mycoplasma pneumoniae pneumonia or Infectious Mononucleosis (IM) -acute secondary optimally reacts at 4°C but also reacts at between 25°C and 30°C The antibody is usually an IgM immunoglobulin which quite efficiently activates complement. Chronic can cause long term anemia 50 years or older is the common age group Antibody specificity is almost always anti-I, less commonly anti-i, and rarely anti-Pr
Laboratory findings in CHD
reticulocytosis and a positive DAT due to complement only The peripheral smear may show agglutinated RBCs, polychromasia, mild to moderate anisocytosis, and poikilocytosis
Immune hemolytic anemia
shortened RBC survival mediated through the immune response, specifically by humoral antibody Three broad categories ~Alloimmune ~Autoimmune hemolytic anemia (AIHA) ~Drug-induced immune hemolytic anemia (DIIHA)
Drug-induced immune hemolytic anemia (DIIHA)
the antibody is formed due to a drug the patient may be taking such as Cephalosporins (a class of antibiotics), most common cause Methyldopa
Alloimmune
the antibody is formed to an antigen introduced to the patient from a foreign blood sample
Autoimmune hemolytic anemia (AIHA)
the antibody is produced to an antigen on the patients RBC's A positive DAT in an anemic patient who has not been recently transfused is suggestive of AIHA
extramedullary hematopoiesis
when RBC production occurs outside of the bone marrow