1. The inheritance of this null leads to a syndrome with hematologic and chemical abnormalities.

The
syndrome includes a mild compensated anemia, reticulocytosis and stomatocytosis. Haptoglobin is
decreased and bilirubin is increased, as well. The null can have two origins, regulator and amorphic.

 Rhnull phenotype

Rhnull syndrome is rare, and is characterized by a complete lack of all Rh antigens. It is caused by
either a mutation in the gene for the Rh-related Antigen (RHAG) (“regulator” type) or a mutation in
the RHCE genes along with a deletion in the RHD gene (“amorphic” type). Rh proteins are essential
parts of the red cell membrane (passing through the membrane 12 times). The absence of the Rh
proteins leads to an alteration of the RBC lipid bilayer, causing the abnormal laboratory results. Quick
tip: There aren’t many things associated with stomatocytes other than Rh null and hereditary
stomatocytosis, so use the Rhnull-stomatocyte association as a key memory fact to store into your
wiring when studying this syndrome.

2. Inheriting this null for the common antigens in the corresponding blood group system leads to a
resistance to the malaria parasite, Plasmodium vivax.

 Fy(a-b-) phenotype

Red blood cells with the Fy(a-b-) phenotype are resistant to invasion by Plasmodium vivax merozoites.
The null is the result of a homozygous inheritance of the silent Duffy allele sometimes called “FY” or
“Fy” (which is actually an altered FY*B allele). The FyFy genotype that leads to the Fy(a-b-) phenotype
is extremely common in West Africa and is found in 68% of African-Americans.

3. The rarest of all blood types is characterized by the absence of the common H antigen. This leads
to the production of a naturally occurring, hemolytic anti-H. People with this null can only be
transfused with red blood cells from other people with this null.

 Bombay phenotype

The Bombay phenotype, Oh, can also be written as “H-.” The homozygous inheritance of the h allele,
hh, prevents a fucose sugar from being added to the precursor structure, paragloboside, on the red
cell surface. This fucose-paragloboside structure is the H antigen. The lack of the fucose prevents
the ABO genes from adding their sugars and creating the regular ABO blood types. Those with the
Bombay phenotype also lack active “secretor” alleles (their genoytpe is sese), and as a result, they
also cannot produce H antigen in secretions or plasma. All Bombay cells will type as group O using
routine testing. The patient plasma will be incompatible with all antibody screening and panel cells.
The only RBCs that will be compatible with the patient will be those from others with the Bombay
phenotype.

4. This null produces red blood cells that are resistant to lysis by the addition of 2M Urea, allowing
for donor compatibility screening for this phenotype without using antisera.

 Jk(a-b-) phenotype
The rare Kidd null phenotype is caused by the inheritance of two mutant, silent alleles at the JK locus
(there are multiple mutant alleles that lead to a lack of Kidd antigen expression). This genotype
produces no Kidd antigens on the red blood cells, and these patients may form anti-Jka, anti-Jkb, and
anti-Jk3 (an antigen present when either Jka or Jkb is present). The Kidd glycoprotein has a specific
function: Transportation of urea across the red blood cell membrane (in fact, the Kidd gene, SLC14A1,
was formerly known as “Human Urea Transport Gene 11” or “HUT11”). Normal RBCs are lysed rapidly
in the presence of 2M urea (a pretty high concentration), because the urea is transported quickly
across the cell membrane, water follows because the cell becomes hypertonic, and the RBC explodes
due to the excess volume. Jk(a-b-) RBCs can’t transport the urea nearly as quickly, however, so they
do not lyse until 30 minutes or more have elapsed. Reference labs can use this fact to quickly screen
for Jk(a-b-), Jk3 negative RBCs by adding 2M urea to multiple samples of donor cells (though they
don’t do this all that often anymore). RBCs that do not lyse can then be confirmed as negative by
serologic or molecular techniques, thus saving time, expense, and potentially scarce reagents.

5. Patients with this rare produce a naturally occurring antibody formerly called “anti-Tja.” This
antibody is actually a combination of three antibodies against three separate antigens in two
different blood group systems. This antibody also has an association with miscarriages early in a
pregnancy.

 p phenotype

The p (“little p”) phenotype is the rarest of five possible phenotypes in the P1PK and GLOB blood
group systems. This phenotype does not produce any of the three main antigens of these systems: P,
P1 or Pk. The antibody originally known as anti-Tja is now known as “anti-PP1Pk.” This is actually three
separable antibodies that will agglutinate red blood cells that are positive for any of those three
antigens. The placenta and fetus contain a large amount of P and P k antigens. Anti-Tja (being IgG) can
damage the placenta and cause fetal demise in the first trimester of pregnancy as a result. Don’t be
confused: Anti-PP1Pk does not typically cause hemolytic disease of the newborn (HDFN)! Instead, the
fetus is harmed indirectly through the antibody attack on the placenta.

6. This null phenotype is found in a blood group system that is phenotypically linked to the secretor-
status of the patient, and has antigens formed in body fluids such as saliva. Name that null!

 Le(a-b-) phenotype

The Lewis null phenotype is not that rare, as 22% of African-Americans and 6% of Caucasians are
Le(a-b-). The rare phenotype in the Lewis system is actually the one where the two main antigens are
both positive: Le(a+b+). The two most common phenotypes, Le(a+b) and Le(a-b+), reveal the secretor
status of the patient, without any need to conduct a secretor test. A patient that is Le(a+b-) is a non-
secretor while a patient that is Le(a-b+) is a secretors. An Le(a-b-) patient lacks an active LE allele
(FUT3), which is also described as the lele genotype. Such patients could be either non-secretors or
secretors, and the secretor test that is performed on a saliva sample from the patient is positive in
approximately 80% of these individuals.

7. Treating red blood cells with a sulfhydryl reagent such as DTT will artificially create red blood
cells of this null, without the normally rare recessive genetic background origin.
  Kell null (K0) phenotype

Kell null (K0) red blood cells can be created in the antibody identification laboratory by treating the
RBCs with a sulfhydryl reagent such as dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or 2-
aminoethylisothiouronium bromide (AET). These reagents destroy the disulfide bonds that assist in
antigen expression in the Kell blood group system. These Kell null cells can be used to identify an
antibody against a high incidence antigen in the Kell blood group system, such as Anti-Kpb.

8. A blood group system may have a null phenotype for multiple genetic reasons. One system has a null
that famously can have three possible genetic origins, two of which do not actually involve the blood
system genes at all.

 Lu(a-b-) phenotype

The Lutheran null phenotype, Lu(a-b-), has three possible genetic origins. The true null is the rarely
seen, autosomal recessive inheritance of two “null” or silent LU alleles, (sometimes written as “LuLu“),
with no resultant Lutheran antigens on the red blood cells. People with this version of the null
phenotype can produce antibodies against all Lutheran blood group system antigens, including the rare
anti-Lu3. The Lu(a-b-) phenotype is also produced by one of two suppressor genes that are not a part
of the Lutheran system at all. The autosomal dominant suppressor gene is called KLF1, and inheritance
of just one of these alleles leads to the “In(Lu) phenotype.” This suppressor gene greatly limits the
expression of Lutheran antigens on red blood cells. Routine phenotyping in the In(Lu)phenotype
detects no Lutheran RBC antigens, but adsorption/elution techniques confirm the weak expression of
the antigens. Because these people have normal Lutheran genes, just weakened Lutheran antigens,
they do not produce Lutheran antibodies except to those antigens to which they truly lack. The third
type of Lu(a-b-) phenotype is inherited in an X-linked fashion, through a mutation in the GATA-1 gene.

9. McLeod syndrome is associated with the null in a blood group system that has only one antigen. The
null can also result in a form of chronic granulomatous disease in males.

 Kx system

Kx blood group system has only one antigen, (stunningly, that antigen is called “Kx”). The antigen
assists in anchoring the antigens in the Kell blood group system to the red blood cell membrane. The
lack of Kx results in the McLeod phenotype, with weakened expression of Kell antigens. The McLeod
syndrome features a compensated hemolytic anemia (classically associated with the presence of
acanthocytes), elevated serum creatinine kinase, and certain neuromuscular disorders. There’s a
pretty good chance that you were tempted to choose “Kell” here, as McLeod is usually taught in
association with the Kell system. However, Kx is in fact a separate blood group system, one whose
lone antigen lives next door to the Kell system antigens on the red cell membrane.

10. This null produces red blood cells that lack the structures Glycophorin A and Glycophorin B and
all antigens located on those structures. This results in the absence of an entire blood group system
in these patients.

 MkMk
Homozygous inheritance of the very rarely seen Mk allele (GYP*01N) will produce red blood cells that
lack Glycophorin A (GPA) and Glycophorin B (GPA). GPA and GPB are the glycoprotein structures that
carry the MNS blood group system antigens, so these patients will not produce any MNS system
antigens such as M, N, S, s, and U. The more famous (and more common) antigen-negative phenotype
in this system is the homozygous inheritance of a null allele for glycophorin B inheritance (GYPB*01N),
leading to a complete lack of GPB and the antigens carried by it (S, s, and U). The S-s-U- phenotype
is seen in roughly 2% of African-Americans. Note: The reason choice D is better than choice C is that
in S-s-U- individuals, the M and N antigens are unaffected, while with M kMk, both M/N AND S/s/U
antigens are absent.

1. Name the three genes responsible for the production of Rh antigens.

 RHAG, RHD, and RHCE

In order to have normal Rh antigens on RBCs, all three genes must be present. RHD and RHCE are
located on chromosome 1. RHD codes for the presence or absence of the D antigen. RHCE has 4 main
alleles to cover the inheritance of C/c and E/e: RHCe, RHCE, RHce, and RHcE (Look closer! The
difference is just the 4 combinations of C or c and E or e). RHAG (Rh associated glycoprotein) is
located on chromosome 6. The presence of this gene allows the proteins resulting from RHD and
RHCE to be incorporated into the RBC membrane. The absence of RHAG can result in a rare condition
known as “Rhnull,” in which the patient has no Rh antigens of any type on their RBCs. Rhnull patients will
typically have hemolytic anemia of varying severity, along with displaying unusual RBC shapes known
as “stomatocytes” (“mouth cells”).

2. Which of the following is TRUE regarding the weak D phenotype?

 Was traditionally identified by an indirect antiglobulin test

Weak D (formerly known as “Du“) occurs when a D-positive person has fewer D antigens on their red
blood cells than normal. This quantitative problem may cause problems with routine Rh typing, as
testing the RBCs with anti-D either gives either no reaction (i.e., they may appear to be D-negative)
or a reaction that is much weaker than the typical strong reactions we expect. This usually is a result
of mutations affecting (but not eliminating) portions of the D antigen. Weak D red cells that react
negatively with laboratory anti-D (which contains a mixture of IgG and IgM anti-D) are shown to be
D-positive when an indirect antiglobulin test (IAT) is performed (In this setting, the IAT is called a
“weak D test.” See my older video, “Weak in the D’s” for more details). We used to think that most
weak D happened due to inheritance of an allele coding for the C antigen on the opposite chromosome
to a D allele (like choice E, and known as “C in trans”), but specific antigen-weakening mutations far
outweigh the “C in trans” mechanism. Weak D and partial D (where the D antigen has missing and
abnormal parts) may have overlapping features, and cannot be reliably distinguished except by Rh
genotyping. Partial D is a problem because those patients may develop anti-D when transfused D-
positive RBCs, so the distinction is important. A 2015 expert taskforce recommended Rh genotyping
for all serologic weak D patients and pregnant moms, to determine if the person has weak D types 1,
2, or 3. Those types are NOT at risk for developing anti-D from transfusion of D-positive RBCs or
delivering a D-positive baby, while other types should be treated as if they were D-negative.
3. Which of the following red blood cell abnormalities is associated with the Rh null phenotype?

 Stomatocytes

Stomatocytes (“mouth cells”) are associated with Rhnull, which is a complete lack of all Rh antigens
(not just D), caused either by mutations leading to inactive Rh genes or by mutations leading to
defects in RHAG (the associated glycoprotein membrane structure that must be present for Rh
antigens to be expressed). A mild hemolytic anemia is also seen in these patients. Schistocytes are
seen in intravascular hemolysis, spherocytes in extravascular hemolysis, ovalocytes in iron-deficiency
anemia, and acanthocytes in the McLeod syndrome associated with the lack of Kx antigen.

8. A 35 year old O-negative male trauma patient receives a transfusion of two units of O-
positive red blood cells before his blood type is known. After his typing is completed, he is
switched to O-negative and he receives 6 additional type-specific RBC units. He survives and is
transferred to the surgical ICU. Which of the following is TRUE regarding his situation?

 He is unlikely to develop delayed hemolysis

This type of event is not uncommon in trauma transfusion, and giving Rh positive RBCs to males in
these settings is fairly standard in the US. Historically, we would say that D-negative people receiving
D-positive RBC transfusions had a roughly 80% chance of forming anti-D. That statistic was based on
exposure in D-negative healthy people, however, and most patients getting this type of exposure are
far from healthy! Current studies have shown the risk to about 22% in hospitalized patients, which
is still really high, but not close to 80%. It is very unlikely that this man will develop an acute hemolytic
reaction, unless he already has a pre-formed anti-D (from a previous D-positive transfusion). Even a
delayed hemolytic reaction is unlikely in this situation, as the transfused cells will likely no longer be
around by the time any antibody could be formed. So, the final question is whether prevention is
indicated in the form of RhIG. I personally do not think that such an intervention is the greatest
idea, due to the facts that a) A large amount of RhIG would be required (at least 20 vials, which could
be given intravenously), and b) If the RhIG works (coating and resulting in clearance of the D-positive
RBCs from the circulation), you might THEN be dealing with the consequences of hemolysis. I don’t
believe in it, but there are those who feel strongly the other way.
11. A 63 year old male presents with a positive antibody screen and clinical and serologic findings
consistent with warm autoimmune hemolytic anemia. In addition, the reference lab technologist
also identifies an antibody that reacts stronger when the test RBCs are D-positive. The
supervisor suggests the technologist consider anti-LWa in addition to anti-D. Which of the
following TWO techniques will MOST assist in differentiating between these two antibodies?

 Adsorption/elution with D-negative RBCs: Anti-LWa detected, anti-D not detected

There is a somewhat confusing relationship between the Rh and LW (“Landsteiner-Wiener”) systems.

LW antigens (including high frequency LWa and LWab and low frequency LWb) are expressed strongly
on D-positive cells but only weakly on D-negative cells (so weakly, in fact, that it may require an
adsorption and elution of the antibody to prove that an LW antigen is present; choice C). As a result,
anti-D and LW antibodies may look the same (this led to historical confusion of the antigens). Both D
and LW antigens are resistant to enzymes such as ficin or papain, so such treatment will not assist in
distinction between the antibodies (choice A). LW antigens are strongly expressed on both D-positive
and D-negative cord cells (choice B). LW antigens are destroyed by treatment with sulfhydryl
reagents such as 0.2M DTT; by contrast, the D antigen is not impacted by DTT treatment (choice D).
LW antigens are often expressed weakly during pregnancy, while the D antigen remains unchanged
(choice E). LW antibodies, by the way, are seen commonly in this clinical setting (warm autoimmune
hemolytic anemia).