Immunohematology exam 2

do Frozen plasma, platelet concentrates, and cryoprecipitate need to be crossmatched​

no

-Plasma products should be ABO serum compatible​

-Cryoprecipitate and platelet concentrates may not need to be ABO compatible​​

Kell group

K, k, Kpa, Kpb, Jsa, Jsb

k cellano

more than 90% of the population - most people do not make antibodies against this due to possessing k​

K1

-less than 9% of the population - most people make antibodies against this if exposed to K through transfusion or pregnancy​

-very immunogenic (second to the D antigen)

K1 and k cellano are

antithetical

different; not inherited together

Kell antigens have ________ regions on the glycoproteins​

disulfide-bonded

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This makes them sensitive to sulfhydryl reagents - Multiple Myeloma pts​

Kp antigens​

Kpa is a low-frequency antigen (only 2%)​

Kpb is a high-frequency antigen (99.9%)​

Js antigens​

Jsa lower frequency

Jsb is a high-frequency antigen (80% to 100%)​

K0 or Kellnull Phenotype​

K0 individuals can develop anti-Ku (Ku is on RBCs except K0)​

K0 pts must be given Knull blood only, because all Kell phenotypes have Ku antigens​

K0 pts have Ku antibodies that will cause transfusion reactions if given Ku​

kell antibodies

-Immunoglobulin G (IgG)

-RBC stimulated (transfusion or pregnancy) - needs exposure to form antibody​

-Agglutinate best in the indirect antiglobulin test (IAT) -37 incubation and wash​

Associated with hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN) - can cross placenta bc IgG antibody​

No effect when treated with enzymes​

Anti-K (Kel1) is the most common​

- low frequency antigen, high frequency antibody​

Kx Blood Group System

​Individuals who lack Kx antigen may demonstrate RBC abnormalities (McLeod phenotype)​

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Seen in males because it is inherited on the X chromosome ​

McLeod syndrome

RBC abnormalities​

Muscular and neurologic defects​

Increased creatine kinase​

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Associated with chronic granulomatous disease​

Impaired phagocytosis

Duffy Blood Group System​

Antigens are well developed at birth​

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Destroyed by enzymes​

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Fya and Fyb​

Codominant alleles​

Most important for transfusion purposes​

Duffy Antibodies​

Anti-Fya and anti-Fyb antibodies​

IgG​​

Stimulated by transfusion or pregnancy (not a common cause of HDFN)​

Do not react with enzyme-treated RBCs​

Duffy and malaria

Most African Americans are Fy(a-b-)​

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Certain malarial parasites (Plasmodium knowlesi and Plasmodium vivax) will not invade Fya- and Fyb-negative cells​​

Kidd Blood Group

3 antigens: Jka, Jkb, and Jk3​

Jk3 is present whenever Jka and Jkb are present - Most white people have Jk3​

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Kidd null phenotypes: Jk(a-b-)​

Usually seen in individuals from the Far East or Pacific Islands (rare)​

May produce anti-Jk3 antibody​

RBCs are resistant to 2M urea​

Show dosage​

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Enhanced by enzymes​

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KIDS CAN DO ANYTHING!​

Anti-Jka and anti-Jkb antibodies​

IgG​

Clinically significant​

May bind complement​

Implicated in HTRs and HDFN​

Common cause of delayed HTRs​

Detection is aided by enzymes, low-ionic-strength solution (LISS), and polyethylene glycol (PEG)​

Lutheran Blood Group

Weakly expressed on cord blood cells ; can be falsely negative because its not fully developed at birth​

Most are high-incidence antigens; antibodies are rare​

Primary antigens include Lua and Lub​

92.4% Lu(a-b+)​

7.4% Lu(a+b+)​

0.2% Lu(a+b-)​

Lunull phenotype is rare, inherited recessively​

Not affected by enzymes​

Anti Lua

May occur without RBC stimulation ​

Without stimulation (no hx of transfusion or pregnancy), it must be IgM​

Immunoglobulin M (IgM) and IgG​

Reacts best at room temperature​

Shows mixed-field pattern​

Not clinically significant​

Anti Lub

Rare due to high incidence of antigen​

IgG​

Reacts best at antihuman globulin (AHG)​

Shows mixed-field pattern​

Associated with transfusion reactions (clinically significant)​

Lewis Blood Group System​

found in secretions (glycoproteins) and plasma (glycolipids)​

The glycolipids adsorb onto the RBC membrane​

Not expressed on RBCs, just absorbed​

Lewis Antigens​

Lea and Leb are not alleles​

Lewis system depends on Hh, Se, and Le genes​

If the Le gene is inherited, Lea substance is produced​

Le, H, and Se genes must all be inherited to convert Lea to Leb​

Lewis Antibodies

produced by Le(a-b-)​

IgM​

Not clinically significant​

Enzymes enhance anti-Leb reactivity​

Anti-Lea binds complement; may cause hemolysis in vitro​

Neutralization can confirm the presence or eliminate reactions with Lewis antibody​​

I Blood Group System i Antigen​

form on the precursor A, B, and H chains of RBCs​

Newborns have i antigen​

Adults have I antigen​

i antigen (linear) converts to I antigen (branched) as a child matures (about 2 years)​

I antibodies

Cold-reacting, IgM, bind complement​

Not clinically significant​

Usually autoantibody (autoanti-I)​

Alloanti-I is rare​

Reactions are avoided by prewarming​

Reacts as compound antibody​

Often found as an anti-IH​

Stronger agglutination with RBCs having many H sites (O and A2)​

Autoanti-I disease

Mycoplasma pneumoniae​

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Cold hemagglutinin disease

Anti-i disease

Infectious mononucleosis​

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Lymphoproliferative disease​

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P blood group system

P1 and P2 antigens​

P1 phenotypes have P and P1 antigens on RBCs​

P1 antigen is detected in plasma and hydatid cyst fluid​; secreter​

P2 phenotypes have P antigens on RBCs​

P2 phenotypes may produce anti-P​

P, Pk, and LKE antigens are high-frequency antigens​

Anti-P1 ​

Found in P2 individuals​

IgM; enhanced by enzymes​

Non-RBC stimulated​

Can be neutralized by P1 substance

Autoanti-P​

​Associated with cold paroxysmal hemoglobinuria​

IgG (Donath-Landsteiner antibody); a biphasic hemolysin that binds with P1 or P2 cells at low temperatures before the complement is activated​

May appear in children after viral infection

Anti-PP1Pk​

Occurs in individuals with the null phenotype​

Causes hemolysis in vitro​

Clinically significant

M and N

Coded by glycophorin A​

Membrane structure is called sialoglycophorin A​

M and N differ at positions 1 and 5 on glycophorin A (GPA)​

Show dosage​

S, s, U

Coded by glycophorin B​

Membrane structure is called sialoglycophorin B​

S and s differ at position 29​

S has methionine; s has threonine​

U antigen​

Present when S or s is inherited​

Absence of glycophorin B (GPB) would result in S-s-U-​

Anti-M

IgM and IgG​

Variable reactions depend on reagent

Anti-S,s,U

Clinically significant IgG​

Anti-U is rare but can be found in S-s- persons (black population)​

Anti-N

Rare IgM

N-like antibodies found in dialysis patients from formaldehyde-sterilized instruments​

for antigen typing, use _____ cells to catch ______

heterozygous cells to catch weak reactions

for selecting cells to rule out, choose _____ cells to account for ______

homozygous cells to account for dosage

Antibody screens are used for​

Patients needing a transfusion​

Pregnant women - to avoid HDFN​

Patients who have had transfusion reactions​

Blood and plasma donors​

use patient's _______ against reagent _________ to detect unexpected antibodies​ during an antibody screen

plasma or serum; RBCs

Unexpected antibodies are a result of ____________

RBC stimulation (transfusion, hemolytic disease of the fetus and newborn [HDFN])​

clinically significant antibodies

Usually IgG​

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React best at 37° C and during the antihuman globulin (AHG) phase (indirect antiglobulin test [IAT])​

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associated with hemolytic transfusion reactions (HTRs) and HDFN

Performing an Antibody Screen​

Patient's plasma or serum is incubated with screening cells ​

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After incubation, an IAT is performed using AHG reagent​

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This will detect any IgG antibodies​

Screening cells

are single or pooled donor group O cells; however, single-donor vials offer increased sensitivity​

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Group O cells are used so that anti-A and anti-B antibodies will not react​

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Each vial (donor) has been phenotyped for each antigen​

antigram

(2 or 3 cells) will list the antigens present in each vial ​

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A reaction to one or more cells indicates the presence of an atypical antibody​

Autocontrol​

Tests a patient's serum with his or her own RBCs​

Determines if antibody is auto or allo antibody​

If autocontrol is positive, run a DAT (patient cells plus AHG) to detect in vivo coating and see if its IgG or C3​

Potentiators​

enhance an antigen-antibody reaction​

Saline (may only enhance if incubated for a long time)​

Low-ionic-strength solution (LISS): common​

Bovine serum albumin (BSA)​

Polyethylene glycol (PEG) - enhances warm antibodies​

IgM antibodies react at ________ temp

room temperature or during immediate-spin (IS) crossmatching; anti-Lea, anti-Leb, anti-M, anti-N, anti-I, anti-P1 antibodies​

IgG antibodies usually react at

37° C or with AHG​

Panel cells that are heterozygous ________________

should not be crossed out, because the antibody may be too weak to react​

rule of three

To ensure valid results, 3 antigen-positive cells must react and 3 antigen-negative cells must not react with the patient's serum or plasma​

If this does not occur, additional panel cells (selected cells) are used​

Phenotyping the patient​

Another way to confirm the presence of antibody is to determine the patient's phenotype for antigen​

Individuals do not make alloantibodies toward antigens on their own RBCs - can be used to rule antibody out​

Dithiothreitol is often used to denature

Kell antigens​

Enzymes are used to ___________ antibody activity​

eliminate or enhance

Duffy and MNS antigens are destroyed, sex linked

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Rh, Kidd, and Lewis antigens are enhanced ​

if most panel cells are positive, expect___________

alloantibody to a high-frequency antigen

High-Titer, Low-Avidity Antibodies​

Antibodies to high-frequency antigens that react weakly​

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React at AHG phase

Antibodies to ​Low-Frequency Antigens

suspected when the screen is negative and crossmatching is positive​

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Only one reactive cell suggests this type of antibody​

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Common antibodies include​

Anti-Cw, anti-Wra, anti-V, anti-Cob, anti-Bga, anti-Kpa, and anti-Lua antibodies​

Enhancing Weak IgG Antibodies​

increase serum to cell ratio

phenotype pt

incubate longer

select different cell

check dosage on weak reactions

repeat with enhancements like enzymes or peg

Cold Alloantibodies

IgM antibodies that react during IS crossmatching

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Anti-P1, anti-M, and anti-N antibodies have variable reactions​

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To enhance reactions, incubate below room temperature​

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To avoid reactions, neutralization can be performed so that clinically significant antibodies may be detected​

Autoantibodies

usually react with all reagents and self and donor RBCs, regardless of the antigens present​​

The autocontrol and DAT are also positive​

Adsorption techniques are usually performed to remove autoantibodies​​

cold autoantibodies

react during IS crossmatching and have a positive autocontrol and DAT (to C3)​

occur in individuals with a history of mild anemia, Mycoplasma pneumoniae infection, or infectious mononucleosis​

anti-I, anti-H, and anti-IH; "cold panels" can aid in identifying them​

Avoiding Cold ​Autoantibody Reactions​

monospecific IgG AHG reagent rather than a polyspecific reagent​

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DO NOT skip IS crossmatching and testing at 37° C​

Use 22% bovine serum albumin instead of LISS​

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Prewarming all tubes to 37° C

Warm Autoantibodies​

More common than cold autoantibodies​

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Warm autoimmune hemolytic anemia may be idiopathic or the result of a disease or drug​

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Most panel cells and the autocontrol are positive​

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Using 22% albumin decreases reactivity​

directed to the Rh system, especially the e antigen​

Patient will have anti-e antibody and the e antigen​

Rh system is most sensitive; partial or weak expression in all of the Rh system, just like weak D ​

Positive DAT​

elution

To identify antibodies when the DAT is positive, the IgG must be detached by using the elution technique​

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The recovered antibody is called the eluate​​

adsorption

Warm autoantibodies may need to be removed to test for underlying alloantibodies​

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Cells can be pretreated with dithiothreitol or a combination of enzymes and dithiothreitol (ZZAP) to remove any in vivo attached antibodies​

Autoantibodies on RBC, alloantibodies in serum​

Phenotyping RBCs coated with IgG can be a challenge

Treating cells with chloroquine diphosphate will disassociate the IgG from the RBCs without harming the antigens​

Crossmatching

mixing donor rbcs and pt plasma

no agglutination = compatible

agglutination = incompatible

serves as a double check of ABO errors

second means of detecting antibodies

immediate spin crossmatch

when pt has no evidence of antibodies

pt serum + donor rbcs mixed and immediately centrifuged (IS)

indirect agglutination testing crossmatching

all phases (IS, 37, AHG) performed if pt has antibody or history of antibody

-if pt has autoantibody, autoadsorbed serum can be used as it removes autoantibodies

Age of sample

The limit is 3 days; after 72 hrs, need new specimen to determine if pt developed new antibodies​

Repeat Testing of Donor Blood​

Whole blood and RBCs must be retyped to confirm the correct ABO labeling​

ABO testing is performed on all units​

D testing is performed only on D-negative units​

Weak D testing is not required​

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Records are kept for ​5 years​

Antigen-negative units are recommended for the following antibodies:​

ABO​

Rh​

Kell​

Duffy​

Kidd​

S​

s​

massive transfusion

is a total volume exchange of blood within 24 hours​

Four unit of packed RBCs, Four units of FFP, 1 unit of platelets​

Group O, D-negative unit is given in an emergency​

ABO-identical unit is given once the blood group is established in the recipient​

Typing and Screening Procedure​

includes ABO, Rh, and antibody screening​

ordered when a surgical procedure uses less than 1 unit of RBCs​

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Crossmatching is performed ​

The goal of a T/S is to conserve the blood inventory​

Preoperative autologous donation

blood drawn before anticipated date

high risk questions do not apply

no age restriction

volume adjusted for weight of pt

hgb 11 or higher

hct 33 or higher

Infants YOUNGER THAN 4 months OF AGE​

ABO and D typing must be performed​

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Serum testing is not necessary- no antibodies developed unless mother's​

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Antibody screening is performed on the infant's or mother's sample if they need blood donation​

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If antibody is present, antigen-negative units are given​

autologous blood

Pt provides own blood for surgeries - Rare phenotypes, Jehovah's witnesses, concerned pts​

preop- blood drawn before anticipated date

normovolemic-remove units at beginning of surgery and reinfusing them at end of surgery

blood recovery- medical device washes, filters, and concentrates blood during operation

permanent deferral for blood donation

- hx of viral hepatitis

- positive test for hep B surface antigen

-hepatitis b core antigen

-hx of Hep C, HIV, Tcell Lymphotropic virus

-hx of chagas disease

-family hx of CJD

-growth hormones

-IV drug user

deferrals for blood donation

hx of malaria 3 years, travel to endemic area 1 year

leishmaniasis travel to Iraq 1 year

MMR, polio, typhoid, yellow fever vaccine 2 weeks

chickenpox vaccine 4 weeks

hep b vaccine 28 days

tattoos, exposure to hiv, jailtime, blood transfusion, rabies vaccine 12 months

physical requirements for blood donation

female H and H : 12.5 and 38%

male H and H : 13 and 39%

systolic bp 90-180

diastolic 50-100

pulse 50-100

weight >= 100lb

age > 16

phlebotomy

Drawn from the antecubital area​

Site is cleaned with 0.7% aqueous iodophor solution followed by 10% povidone-iodine​

A 16-gauge needle is used​

Allogeneic donations:

donations for the general population​​

directed donations

donations for specific recipients

No evidence supports the theory that directed donations are safer than allogeneic donations​

Apheresis​

components separated, remaining blood returned to donor

-leukapheresis wbc removed

-plateletpheresis

plateletpheresis

no more than twice a week or 24 times a year, at least 48 hours apart

plasmapheresis

Infrequent: no more than once every 4 weeks​

Frequent: immunoglobulin G and M (IgG and IgM) levels are monitored every 4 months if donations are more than once every 4 weeks​

Red cell apheresis

Two units of RBCs may be donated if weight and height requirements are met​

Deferral is 16 weeks following double RBC donation​

required test for donations

rbc antigens - abo and d

clinically significat antibodies - ab screen

hepatitis - HBsAg, anti HCV, anti HBc, HCV NAT

HIV - anti HIV, HIV NAT

HTLV - anti HTLV

syphilis - RPR, hemagglutination

WNV - WNV NAT

Chagas - IgG Ab to T cruzi

Antibody screen test

if significant antibodies are present, plasma and platelets cannot be used

rbc can be used

ELISA

EIA detects antigens or antibodies using a solid object (e.g., plastic bead) coated with antigen or antibody​

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Indirect EIA​

Detects antibodies​

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Sandwich EIA​

Detects antigen​

Nucleic Acid Testing (NAT)​

NAT amplifies nucleic acids of infectious agents and identifies viral RNA​

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NAT can detect very low numbers of viral copies in plasma before antibodies appear​

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Chemiluminescence​

emission of light from a chemical reaction​

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labels are attached to an antigen or antibody where the highest light intensity emitted is measured​

very sensitive and stable with fast tat

Sensitivity

identify infected individuals as positive

100x #of positive/total #infected

Specificity

identify noninfected individuals as negative

100x#of negative/total number of infected

hepatitis testing

HBsAg indicated individual is infectious

Anti HBc appears after surface antigen but before symptoms; pt had HBV in past; test to see if from vaccine or actual pt ab

Anti HCV detectable 10 weeks after infection

human retroviruses

A retrovirus contains reverse transcriptase, which allows the virus to convert RNA to DNA and then integrate the DNA into the cell​

Lentivirus (HIV types 1 and 2)​

Oncornavirus (human T-cell lymphotropic virus [HTLV] types I, II, and V)​

HIV 1 and 2

cause aids

infects CD4 helper t lymphs

antibody develops 22-25 days after infection

HIV NAT 9.1 day window

HTLV 1 and 2

1 - t cell leukemia

2 - large granular lymphocyte leukemia

western blot

Confirmatory test for antibody to HIV types 1 and 2 and HTLV types I and II​

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Antibodies in serum are tested for a reaction with individual protein bands (antigen) on strips​

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Patterns are compared with a strip containing all HIV proteins​

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Two of the following bands must be positive​

p24​

gp41​

gp120/160​

look back investigation

actions taken when donor test results are positive for the hepatitis virus, HIV, HTLV, or WNV​

Quarantine prior to donations from the donor​

Notify facilities that received products​

Donor is further tested​

Destroy (or relabel) prior collections​

Notify transfusion recipients​