| The ISBT terminology for RBC surface antigens provides
a standardized numeric system for naming authenticated
antigens that is suitable for electronic data processing
equipment
True or False | True |
| In the ISBT classification, RBC antigens are assigned
a | six-digit identification number: |
| antigens are not synthesized by the
RBCs. These antigens are adsorbed from plasma onto
the RBC membrane | Lewis |
| The Le gene codes for | L-fucosyltransferase, |
| The Le gene is needed for the expression of Lea substance,
and Le and Se genes are needed to form | Leb
substance. |
| more common among blacks
than among whites and results in the Le(a–b–)
phenotype. | lele genotype |
| antigens are poorly expressed at birth | Lewis |
| Lewis antibodies are generally (Blank) and made by (blank) | IgM (naturally occurring)
made by Le(a–b–) individuals. |
| frequently encountered in pregnant
women. | Lewis antibodies |
| not considered significant for
transfusion medicine. | Lewis antibodies |
| consists of the biochemically
related antigens P, P1, Pk and LKE. | P blood group |
| expression is variable, and poorly expressed at birth | P1 antigen |
| is a common naturally occurring IgM antibody
in the sera of P1– individuals; it is usually
a weak, cold-reactive saline agglutinin and can
be neutralized with soluble P1 substance found in
hydatid cyst fluid. | Anti-P1 |
| is produced by the rare p individuals early
in life without RBC sensitization and reacts with all | Anti-PP1Pk |
| RBCs except those of other p individuals. Antibodies
may be a mixture of | IgG and IgM |
| is associated with
spontaneous abortions. | Anti-PP1Pk |
| is found as a naturally occurring alloantibody
in the sera of Pk individuals and is clinically significant. | Alloanti-P |
| is most often the specificity associated with
the cold-reactive IgG autoantibody in patients with
paroxysmal cold hemoglobinuria (PCH) | Auto-anti P |
| The autoanti-P of PCH usually does not react by routine
tests but is demonstrable as | biphasic hemolysin only
in the Donath-Landsteiner test. |
| Relationship of I and i antigens | antithetical, reciprocal relationship |
| Most adult RBCs are rich in I and have only trace
amounts of i antigen.
True or False | True |
| At birth, infant RBCs are rich in i; I is almost undetectable;
over the next | 18 months , the
infant’s RBCs will convert from i to I antigen. |
| is typically a benign, weak, naturally occurring,
saline-reactive IgM autoagglutinin, usually detectable
only at 4°C | Anti-I |
| typically a strong cold autoagglutinin
that demonstrates high-titer reactivity at 4°C and
reacts over a wide thermal range (up to 30°–32°C). | Pathogenic anti-I |
| may develop
strong cold agglutinins with autoanti-I specificity | M. pneumoniae |
| rare IgM agglutinin that reacts optimally
at 4°C; potent examples may be associated with infectious
mononucleosis. | Anti-i |
| are cold-reactive saline agglutinins
that do not bind complement or react with enzyme treated
cells;may demonstrate dosage effect | Anti-M and Anti-N |
| are IgG antibodies, reactive at 37°C
and the antiglobulin phase. They may bind complement
and have been associated with HDFN and HTRs. | Anti-S and anti-s |
| In MNS blood system, it is usually an IgG antibody and has been associated with HTRs and HDFN. | Anti-U |
| well developed at
birth and are not destroyed by enzymes. | kELL |
| are destroyed by DTT,
ZZAP, and glycine-acid EDTA. | kELL |
| Excluding ABO, the K antigen is rated second only to | D antigen in immunogenicity. |
| antigen is high prevalence. | k |
| is usually an IgG antibody reactive in the
antiglobulin phase and is made in response to pregnancy
or transfusion of RBCs; it has been implicated
in severe HTRs and HDFN. | Anti-K |
| affecting only males, is described
as a rare phenotype with decreased Kell system
antigen expression. | Mcleod phenotype |
| the clinical manifestations of abnormal RBC morphology,
compensated hemolytic anemia, and neurological
and muscular abnormalities. Some males with the | Mcleod Syndrome |
| also have the X-linked chronic
granulomatous disease. | McLeod phenotype |
| antigens are destroyed by enzymes and
ZZAP; they are well developed at birth. | Fya and Fyb |
| BCs resist infection by the malaria organism
P. vivax. | Fy(a–b–) |
| are usually IgG antibodies and
react optimally at the antiglobulin phase of testing;
both antibodies have been implicated in delayed HTRs
and HDFN. | Anti-Fya and anti-Fyb |
| may demonstrate dosage, are
often weak, and found in combination with other
antibodies; both are typically IgG and reactive in the
antiglobulin test. | Anti-Jka and anti-Jkb |
| may bind complement and are
made in response to foreign RBC exposure during
pregnancy or transfusion. | Kidd antibodies |
| antibodies are a common cause of delayed
HTRs. | Kidd system antibodies |
| antibody reactivity is enhanced with
enzymes, LISS, and PEG. | Kidd |
| are antigens produced by codominant
alleles; they are poorly developed at birth. | Lua and Lub |
| may be a naturally occurring saline agglutinin
that reacts optimally at room temperature. | Anti-Lua |
| is usually an IgG antibody reactive at the
antiglobulin phase; it is usually produced in response to
foreign RBC exposure during pregnancy or transfusion. | Anti-Lub |
| phenotype is rare and may result
from three different genetic backgrounds; only individuals
with the recessive type Lu(a–b–) can make
anti-Lu3. | The Lu(a–b–) phenotype |
| are located on a major RBC
protein, band 3, also known as the RBC anion exchanger
(AE1). | The Diego system antigens |
| are generally considered
to be clinically significant; all have caused severe
HTRs and HDFN | Anti-Dia, anti-Dib, and anti-Wra |
| xpression requires the presence of a normal GPA
(MNS system); | Wrb |
| is a fairly common antibody to a highprevalence
antigen that is sometimes clinically significant
and sometimes insignificant | Anti-Yta |
| antigen is found on the short arm of the
X chromosome and is of higher prevalence in females
(89%) than in males (66%). | Xga |
| are rare and
little is known about their clinical significance, has been observed in
the Marshall Islands and New Guinea. | Scianna system antigens |
| the antigens are carried on aquaporin 1, a red
cell water channel | Colton system |
| has a phenotypic relationship with the D antigen | LW |
| reacts strongly with D+ RBCs and can look
like anti-D. | Anti-LW |
| are located
on the complement fragments C4B and C4A, respectively,
that are adsorbed onto RBCs from plasma. | Chido/Rodgers system |
| react
weakly, often to moderate or high-titer endpoints in
the antiglobulin test and may be tentatively identified
by plasma inhibition methods. | anti-Ch and anti-Rg |
| are very rare outside of
Papua, New Guinea. | Gerbich-negative phenotypes |
| are carried on the decay accelerating
factor and are distributed in body fluids and on
RBCs, WBCs, platelets, and placental tissue. | Cromer antigens |
| are located on complement
receptor 1 (CR1)
have weak and “nebulous” reactivity at
the antiglobulin phase; they are not inhibited by plasma. | Knops antigens |
| more prevalent in Arab and Iranian
populations, | Ina antigen |
| found more commonly in Japanese, but
clinical significance is not well established, since it
is a rare antibody; it has caused HTRs and a fatal case
of HDFN | Anti-Jra |
| has characteristic shiny and refractile agglutinates
under the microscope and is inhibited with
urine from Sd(a+) individuals. | Anti-Sda |
| 1. The following phenotypes are written incorrectly
except for:
a. Jka+
b. Jka+
c. Jka(+)
d. Jk(a+) | d |
| 2. Which of the following characteristics best describes
Lewis antibodies?
a. IgM, naturally occurring, cause HDFN
b. IgM, naturally occurring, do not cause HDFN
c. IgG, in vitro hemolysis, cause hemolytic transfusion
reactions
d. IgG, in vitro hemolysis, do not cause hemolytic transfusion
reactions | b |
| 3. The Le gene codes for a specific glycosyltransferase that
transfers a fucose to the N-acetylglucosamine on:
a. Type 1 precursor chain.
b. Type 2 precursor chain.
c. Types 1 and 2 precursor chain.
d. Either type 1 or type 2 in any one individual but not
both. | a |
| 4. What substances would be found in the saliva of a
group B secretor who also has Lele genes?
a. H, Lea
b. H, B, Lea
c. H, B, Lea, Leb
d. H, B, Leb | c |
| 5. Transformation to Leb phenotype after birth may be as
follows:
a. Le(a–b–) to Le(a+b–) to Le(a+b+) to Le(a–b+)
b. Le(a+b–) to Le(a–b–) to Le(a–b+) to Le(a+b+)
c. Le(a–b+) to Le(a+b–) to Le(a+b+) to Le(a–b–)
d. Le(a+b+) to Le(a+b–) to Le(a–b–) to Le(a–b+) | a |
| 6. In what way do the Lewis antigens change during
pregnancy?
a. Lea antigen increases only
b. Leb antigen increases only
c. Lea and Leb both increase
d. Lea and Leb both decrease | d |
| A type 1 chain has:
a. The terminal galactose in a 1-3 linkage to subterminal
N-acetylglucosamine.
b. The terminal galactose in a 1-4 linkage to subterminal
N-acetylglucosamine.
c. The terminal galactose in a 1-3 linkage to subterminal
N-acetylgalactosamine.
d. The terminal galactose in a 1-4 linkage to subterminal
N-acetylgalactosamine. | a |
| 8. Which of the following best describes Lewis antigens?
a. The antigens are integral membrane glycolipids
b. Lea and Leb are antithetical antigens
c. The Le(a+b–) phenotype is found in secretors
d. None of the above | d |
| 9. Which of the following genotypes would explain RBCs
typed as group A Le(a+b–)?
a. A/O Lele HH Sese
b. A/A Lele HH sese
c. A/O LeLe hh SeSe
d. A/A LeLe hh sese | b |
| 10. Anti-LebH will not react or will react more weakly with
which of the following RBCs?
a. Group O Le(b+)
b. Group A2 Le(b+)
c. Group A1 Le(b+)
d. None of the above | c |
| 11. Which of the following best describes MN antigens and
antibodies?
a. Well developed at birth, susceptible to enzymes,
generally saline reactive
b. Not well developed at birth, susceptible to enzymes,
generally saline reactive
c. Well developed at birth, not susceptible to enzymes,
generally saline reactive
d. Well developed at birth, susceptible to enzymes,
generally antiglobulin reactive | a |
| 12. Which autoantibody specificity is found in patients with
paroxysmal cold hemoglobinuria?
a. Anti-I
b. Anti-i
c. Anti-P
d. Anti-P1 | c |
| 13. Which of the following is the most common antibody
seen in the blood bank after ABO and Rh antibodies?
a. Anti-Fya
b. Anti-k
c. Anti-Jsa
d. Anti-K | d |
| 14. Which blood group system is associated with resistance
to P. vivax malaria?
a. P
b. Kell
c. Duffy
d. Kidd | c |
| 15. The null Ko RBC can be artificially prepared by which
of the following treatments?
a. Ficin and DTT
b. Ficin and glycine-acid EDTA
c. DTT and glycine-acid EDTA
d. Glycine-acid EDTA and sialidase | c |
| 16. Which antibody does not fit with the others with respect
to optimum phase of reactivity?
a. Anti-S
b. Anti-P1
c. Anti-Fya
d. Anti-Jkb | b |
| 17. Which of the following Duffy phenotypes is prevalent
in blacks but virtually nonexistent in whites?
a. Fy(a+b+)
b. Fy(a–b+)
c. Fy(a–b–)
d. Fy(a+b–) | c |
| 18. Antibody detection cells will not routinely detect which
antibody specificity?
a. Anti-M
b. Anti-Kpa
c. Anti-Fya
d. Anti-Lub | b |
| 19. Antibodies to antigens in which of the following blood
groups are known for showing dosage?
a. I
b. P
c. Kidd
d. Lutheran | c |
| 20. Which antibody is most commonly associated with
delayed hemolytic transfusion reactions?
a. Anti-s
b. Anti-k
c. Anti-Lua
d. Anti-Jka | d |
| 21. Anti-U will not react with which of the following RBCs?
a. M+N+S+s–
b. M+N–S–s–
c. M–N+S–s+
d. M+N–S+s+ | b |
| 22. A patient with an M. pneumoniae infection will most
likely develop a cold autoantibody with specificity to
which antigen?
a. I
b. i
c. P
d. P1 | a |
| 23. Which antigen is destroyed by enzymes?
a. P1
b. Jsa
c. Fya
d. Jka | c |
| 24. The antibody to this high-prevalence antigen demonstratesmixed-field agglutination that appears shiny andrefractile under the microscope:a. Velb. JMHc. Jrad. Sda | d |
| 25. Which of the following has been associated with causing
severe immediate HTRs?
a. Anti-JMH
b. Anti-Lub
c. Anti-Vel
d. Anti-Sda | c |
| 26. Which of the following antibodies would more likely be
found in a black patient?
a. Anti-Cra
b. Anti-Ata
c. Anti-Hy
d. All of the above | d |
| 27. Which of the following antigens is not in a blood group
system?
a. Doa
b. Vel
c. JMH
d. Kx | b |
| 28. A weakly reactive antibody with a titer of 128 is neutralized
by plasma. Which of the following could be the
specificity?
a. Anti-JMH
b. Anti-Ch
c. Anti-Kna
d. Anti-Kpa | b |
| 29. An antibody reacted with untreated RBCs and DTTtreated
RBCs but not with ficin-treated RBCs. Which of
the following antibodies could explain this pattern of
reactivity?
a. Anti-JMH
b. Anti-Yta
c. Anti-Kpb
d. Anti-Ch | d |
| 30. The following antibodies are generally considered clinically
insignificant because they have not been associated
with causing increased destruction of RBCs, HDFN,
or HTRs.
a. Anti-Doa and anti-Coa
b. Anti-Ge3 and anti-Wra
c. Anti-Ch and anti-Kna
d. Anti-Dib and anti-Yt | c |
| Lewis antibodies are increased in | Pregnancy |
| It can be mostly seen in patients with renal disease who undegoes dialysis on equiptment sterilized by formaldehyde | Anti-N |
| Strong-Cord RBCS
Weak- Normal Adult RBC
Most weak- i | I (t) |
| Associated with Human Leukocyte Antigen and can be destroyed by enzyme | Bennet Goodspeed |
| Characteristics of Anti-i except: | IgM in nature |
| The only system not synthesized by RBC | Lewis |
| The following are characteristics of Anti-Lea except: | Uncommon antibody in the Lewis system |
| Lewis antigen can be detected in plasma on: | After 10 days of life |
| Antigen in this system can be expressed as early as 12 weeks, weaken gestational age and fully developed at 7 years | P blood system |
| The following are characteristics of anti-M except | Common in Children than in Adults and in patients with Burns |
| Associated with Paroxysmal Cold Hemoglobinuria | Auto-anti P |
| Patients with this antigen has a resistance to infections such as Malaria | Duffy |
| The following are characteristics of anti-P1 excepts: | Optimally reactive at 37 |
| K antigen can be detected at | 10 weeks of fetal life |
| The following are characteristics of Anti-Lua except: | Reacts at AHG phase |
| pH requirement of anti-M | 6.5 |
| True Lewis phenotype who inherits Le and Se can be expressed | 7 years of life |
| The following are characteristics of S and s antigen except | Cannot be detected in platelets lymphocytes and monocytes |
| Associated with Delayed Hemolytic Transfusion reaction | Kidd |
| Can be detected on renal endothelium and epithelium | MNS |
| The antibody of these blood group demonstrate loose mixed-field agglutination pattern with an optimal pH of 12 - 23 C | Lutheran |
| Component of glycoprotein for the expression of Lewis | 80% Carbo
15% amino |
| Phenotype of I at 18 months of life | I |
| A phenotype can be expressed if there is no production of GPA but GPB is not affected | En(a-) |
| Rated next to D in immunogenicity can be found in the RBCs and platelets | Kell |
| The following can demonstrate dosage effect except | Anti-M |
| The true lewis phenotype of a person who inherited the following gene : Le and sese | Le (a+b-) |
| • The focus of antibody detection is to detect | irregular” or “unexpected” antibodies of the ABO system |
| The unexpected antibodies with primary importance are; | 1. Immune Alloantibodies
- which are produced in response to red blood cell (RBCs) stimulation through transfusion, transplantation, or pregnancy.
2. Naturally Occurring Antibodies
- may form as a result of exposure to environmental sources, such as pollen, fungus, and bacteria, which have structures similar to some RBC antigens.
3. Passively Acquired Antibodies
- Antibodies produced in one individual and then transmitted to another via plasma-containing blood components or derivatives such as intravenous immunoglobulin (IVIG) |
| • Clinically Significant alloantibodies | - Those that can cause decrease survival of the RBCs
- IgG antibodies that react at 37C or at AHG (Antihuman globulin phase) of the Antiglobulin Test (IAT)
Note: When unexpected antibody is detected in the Antibody screening, an Antibody detection is performed so that appropriate transfusion considerations put in place. |
| can complicate detection | • Autoantibodies |
| • Major Blood Groups: | Kell, Duffy, Kidd, MNSs, P, and I |
| • Minor Blood Groups | Diego, Cartwright, Xg, Scianna, Dombrock, Colton, Chido-Rodgers, Gerbich, Cromer, Knops, Indian, JMH |
| • To detect clinically significant antibodies | Antibody screening |
| requires the use of an antibody screen to detect clinically significant antibodies in both the blood donor and the intended recipient as part of pretransfusion compatibility testing | - The AABBs Standard for Blood Banks and Transfusion Services |
| • Patient’s serum/plasma is tested against RBCs with known antigen, traditional testing | Tube technique |
| AHG reagents | used to sensitize the reagent RBCs with the patient’s antibodies, followed by formation of visible RBC agglutinates |
| 1. Immediate spin phase: cold reacting Ab | It is to detect antibodies reacting at room temperature.
Not required and may lead to the detection of Cold reacting antibodies.
IgM |
| 2. 37C incubation phase: warm reacting Ab | IgG antibodies is present in patients Serum, sensitize any RBCs that possess the target antigen, coating those RBCs with antibody. |
| may be added before incubation to 37C to increase the degree of sensitization. | Enhancement medium |
| To observe for hemolysis, | carefully removed from the centrifuge so as not to dislodge the RBC button. The supernatant is observed for pink or red discoloration.
May appear as a loss of cell button mass |
| Tubes only need to be observed for hemolysis when | fresh serum and reagent RBCs that to not contain EDTA |
| 4. Observe agglutination | the tube is gently tilted or rolled to dislodge the cell button. |
| 5. Washing phase (3x) | The tubes are washed with 0.9% saline a minimum of 3 times to remove all antibodies that remain unbound.
7.2 – 7.4 pH saline |
| 6. AHG / Coomb’s serum phase: non-agglutinating warm reactive antibodies | The tubes are centrifuged and examined for hemolysis and agglutination |
| 7. Addition of Coombs control cells | All negative tests will have Coombs’ control cells (also known as check cells) added to confirm the negative result |
| Rbc reagents are from the | Group O |
| • Homozygous | From an individual who inherited only ONE allele at a given locus
“Double dose” of antigen (Many antigen)
Ideal one
Dosage effect- has strong reaction |
| • Heterozygous | Inherited TWO different allele at a locus
“Share” available antigen
The expression is not strong |
| may be added to the cell/serum mixture before the 37°C incubation phase to increase the sensitivity of the test system. These reagents may also allow for a shortened incubation time | • Enhancement reagents, or potentiators |
| Normal Incubation time | 45 minutes to 1 hour) |
| 22% Albumin | • to produce an ionic cloud around each RBC, forcing the cells apart.
• Reduce zeta potential
• Disperse charges thus allowing the RBCs to approach each other
• Increase the chances of agglutination |
| What is zeta potential | difference in electrical potential between the surface of the RBC and the outer layer of the ionic cloud |
| Low Ionic Strength | • Contains glycine in albumin solute
• Reduce Zeta potential
• Increase the uptake of Ab into the RBC during the sensitization phase
• Increase the possibility agglutination |
| Polyethylene Glycol (PeG) | • Removes water from the test system
• Increases the degree of RBC sensitization
• Can cause nonspecific aggregation of cells, so centrifugation after the 37°C incubation is not performed
• More sensitive than Albumin, LISS, or Saline |
| Peg is not appropriate to use in | in patients with elevated levels of plasma protein, such as in multiple myeloma, PEG is not appropriate for use due to increased precipitation of proteins because of the presence of Glycol |
| Ahg reagents | • Allows for the agglutination of incomplete antibodies
• Contains anti-IgG to react on antibodies that are IgG in form
• Presence of anticomplement can lead to the detection of Clinically significant antibodies. |
| Two types | Polyspecific
Monospecific |
| • Polyspecific AHG (Polyvalent or broad-spectrum Coombs’ serum) | Contains antibodies to both IgG and complement (can detect IgM)
Antibody to C3 component (CD3) are more desirable in the reagent, as these are more abundant on the RBC surface during complement activation and lead to fewer false-positive reactions. |
| • Monospecific AHG | Contains Anti-IgG only
Commonly used in donor antibody screening and pretransfusion testing in the recipient.
Avoid time consuming investigations |
| Any test that is negative after adding the AHG reagent should be controlled by adding | Coombs’ control cells |
| prove that:
Adequate washing is performed to remove unbound antibodies.
AHG reagent was added
Reagent was working properly | • Coombs control cells (Check Cells) |
| Ideal result of Coombs Check cells | Positive |
| • Microtube filled with a dextran acrylamide gel | Gell technique |
| Gel technique uses | LISS to a concentration of 0.8% |
| Gel technique specimen and reagent | • Specimen: Patients Serum / Plasma
• Reagent: Screen cells |
| How many chambers in gel technique | There are up to six chamber/gel microtubules contained in a plastic card, about the size of a credit card. The procedure is much simpler |
| Procedure | 1. Incubate at 37C for 15 minutes – 1 hour to allow the sensitization to occur. (Depends on the type of Gel)
2. Centrifuge for 10 minutes, RBCs are forced out of the reaction chamber down into the gel.
The gel contains Anti-IgG
If sensitization occurred, the anti-IgG will react with the antibody-coated RBCs, resulting in agglutination |
| Gel technique | (+) Agglutination: Trapped with the gel. (Proceed to Antibody Identification)
(-) Agglutination: Pellet at the bottom of the microtubule. (The RBCs will freely settle down in the bottom)
Note: The reaction is opposite to the tube method |
| • Commonly used to perform the antibody screen
• Uses Microtiter wells | Solid phase technique |
| Example of Solid Phase | Immucor’s Capture-R |
| Procedure | 1. Patients’ serum/plasma added to each well in the screen cell set along with LISS.
2. Incubate at 37C to allows any antibodies present to react with the antigens (Sensitization)
3. Washing of the well to remove unbound antibodies
4. Adding indicator cells (coated with Anti-IgG, to allow reaction to occur)
5. Centrifugation for several minutes |
| Interpretation | (+) Sensitization: Diffuse pattern (4+)
(-) Sensitization: Pellet at the bottom of the well |
| sample from the patient and donor | Crossmatching |
| there is a reagent specific for the antibody to react to check the antibodies present. | Antibody detection |
| Interpretation: | Positive or there is agglutination= presence of antibodies |
| 1. In what phase(s) did the reaction(s) occur? | IgM Reacts best at room temp or lower Cause agglutination in the Immediate Spin Reaction IgG Reacts best at AHG phase May be IgG, IgM or mixture or both Lewis and M antibodies |
| 2. Is the autologous control negative or positive? | • Autologous Control- the patient’s RBCs tested against the patient’s serum or plasma in the same manner as the antibody screen
• (+) Ab screen and (-) Autologous control
Alloantibody detected
• (+) Autologous control- may be caused by alloantibody coating circulating donor RBCs.
Autoantibody detected due to:
Recent Transfusion in the previous 3 months
Require time and experience |
| 3. Did more than one screen cell sample react? If so, did they react at the same strength and phase | Single Antibody - screen cells yielding a positive reaction react at the same phase and strength.
Multiple Antibodies - when screen cells react at different phases or strengths
Autoantibodies - when the autologous control is positive. |
| 4. Is hemolysis or mixed-field agglutination present? | Mixed Field Agglutination Anti-Lea Anti-Leb Anti-PP1Pk Anti-Sda Lutheran Abs |
| 5. Are the cells truly agglutinated, or is rouleaux present? | • Rouleaux
Not a significant finding but is easily confused with antibody-mediated agglutination
Altered albumin to globulin ratio (patients with multiple myeloma)
High molecular weight plasma expanders (Dextran) |
| Characteristics of Rouleaux | Stacked coin” appearance when viewed microscopically
Observed in all tests containing the patient’s serum, including the autologous control and the reverse ABO grouping.
Does not interfere with the AHG phase of testing because the patient’s serum is washed away prior to the addition of the AHG reagent.
Unlike agglutination, rouleaux is dispersed by adding one to three drops of saline to the test tube.
Confirm in the microscope |
| Limitations | 3 Cells Screen- to identify if there is presence of antibodies, if it gives positive reactions then it means that you are 95% confident
Note: If the titer is very low the antibody cannot be detected |
| Factors that may influence the sensitivity of the antibody screen: | 1. Cell-to-Serum Ratio · Whenantibody is present in the test system in excess false-negativereactions occur as a result of prozone. · Whenthe antigen is in excess, false-negative reactions occur due to postzone · Aratio of two drops of serum to one drop of the RBC suspension typicallygives the proper balance between antigen and antibody to allowsensitization and agglutination to occur. Note: When an antibodyis weak, the amount of serum in the test system may be increasedto four to ten drops if NO potentiators (tube system), providing moreantibodies to react with the available antigens. 2. Temperature and Phaseof Reactivity · Omitthe immediate spin and room temperature phases to limit the detection ofinsignificant cold antibodies. · Spinand RT phase 3. Length of Incubation · Iftoo little contact time is allowed, too few RBCs will be sensitized · Ifthe incubation time is allowed to continue for too long, bound antibody maybegin to dissociate from the RBCs. · Asaline environment may require an incubation time of 30 minutesto 1 hour · Potentiators mayshorten the incubation time to as little as 10 minutes. 4. Ph · Mostantibodies react best at a neutral pH between 6.8 and 7.2 · Anti-Mdemonstrate enhanced reactivity at a pH of 6.5 · Acidifyingthe test system may aid in distinguishing anti-Mfrom other antibodies |
| • Used to identify clinically significant of IgG | Antibody Identification |
| Patient History | • Age, sex, race, diagnosis
• Transfusion and pregnancy history
Anti-M, leb
• Medication and intravenous solution
IVIG, RhIG, Antilymphocyte Globulin
Passively transfer Ab (Anti-A, Anti-B, Anti-D)
Results: presence of unexpected serum antibody |
| Reagents | • Panel: collection of 11-20 group “O” cells with various Ag expression |
| Procedure | 1. Immediate spin
Drops (px serum) + 1 drop (panel cells)
2. Incubation Phase
2 drops of LISS
Incubate for 10-15 mins
3. AHG phase
Wash the red cell (3X)
2 drops of AHG after washing
4. Check Cells
Confirms true negative |
| also known as inhibition, addition of substance in the body that in nature has antigenic structures similar to RBC antigens. It allows separation. | Neutralization |
| Remove and recover antibody from red cells by techniques such as glycine acid, organic solvents | 2. Elution |
| • Proteases: | Ficin (figs), papain (papaya)
Trypsin (calfspleen) Bromelin (pineapple) |
| used to remove sialic acid | • Neuraminidase |
| Add enzyme directly to serum/cell mixture | One-stage procedure |
| Enzyme solution +test red cell @37 degree Celsius
Wash to remove enzyme completely
Retest cells with serum being investigated | Two-stage procedure (preferred) |
| Destroyed | Duffy, Xg, JMH, Ch/Rg, Pr, Mn |
| Enhanced | Lewis, li, P, kidd, ABO, Rh |
| Unaffected | Kell |
| Variable | Se, lutheran |
| used to create RBC negative for all antigens of the kell BGS (except Kx) | AET and DTT |
| Removal of autoantibodies from patient cells provides free antigen sites for adsorption of autoantibody from the serum | 5. ZZAP |
| Destroy kell, cartwright, gerbich, Dombrock | 5. ZZAP |
| Washed packed red cells are incubated in a 1:4 ratio with chloroquine at room temperature
Dissociates IgG from patient red cells with a positive DAT so that they may be typed with blood grouping reagents that require an indirect antiglobulin technique
Reagent is removed by multiple saline washes
Only anti-IgG should be used as the AHG reagent, as complement | 6. Chloroquine diphosphate |
| Useful kin immunohematologic testing | 7. Lectins (plants) and proteins (snails) |
| Ulex europaeus | a-i-fucose |
| Vicia graminea | O-linked N blood group tetrasaccharides (galactose (B1,3) N-acetyl-D-galactosamine) |
