Download PART FOUR (4) Transfusion medicine

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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
PART FOUR (4)
Transfusion medicine
Clinical Laboratory Diagnosis
For
General Clinical Laboratory Scientists
Transfusion Medicine
By
Kourosh Teymourian, CLS
Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
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Contents
Chapter One (1)
1.
2.
3.
4.
5.
6.
Blood Collection and Processing and Donor selection.
Rh typing
ABO typing
Detection of unexpected antibodies
Various types of blood donations
Adverse reaction & Reaction grades at voluntary blood donation
Chapter Two (2)
1.
2.
3.
4.
5.
6.
7.
8.
9.
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Definition and types of hemapheresis
Whole blood
Blood components
Red Blood Cell components
Platelet components
Granulocytes
Fresh Frozen plasma
Cryoprecipitate anti-hemophilic Factor
Clotting Factor Concentrates
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10. ABO/ABH blood groups system
11. Rh Blood Group System
12. Other Blood Group System
13. Lewis system
14. I & i blood Group System
15. P system
16. Kell System
17. MNSs
18. Duffy system
19. Lutheran system
20. Kidd Blood Group System
21. Other antigens
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Chapter Three (3)
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1. DAT (Direct Anti-globulin Test)
2. IAT (Indirect Anti-globulin Test)
3. Pre-transfusion testing
4. Type & screen
5. Types of component transfusion
6. Autologous type
7. Preoperative
8. Intraoperative
9. Postoperative
10. Post transfusion reactions
11. Side effects and adversities of transfusion
12. Disease transmitted through transfusion
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Chapter Four (4)
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1)
2)
3)
4)
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HDN/Autoimmune Hemolytic anemia (warn type)
CHD/Cold Hemoagglutinin disease
Proxysmal Cold Hemoglobinuria (PCH)
Drug induced hemolytic anemia
References
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Normal Values
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Chapter One (1)
Blood collection, processing and donor selection
(Rodgers GP et
al/Quinley DE et al))
T
he first aspect of blood transfusion is the determination and administration of a
safe blood unit to the recipient, this is done by a good clinical history of the
donor(s), Quality Assessment/Assurance and Quality Control practice, and some
preliminary testing such as hemoglobin, hematocrit, blood pressure and
inquisition of any clinical history and background check (delta checks) of clinical illness,
if any that might affect the outcome of the transfusion and blood banking. Donor
registration always starts by a proper identification of the patient is a now a days
facilitated by bar coding and some systems use the applicability of a patients wrist band
that can identify donor blood group and transmit it to the computer-attached with
software applications.
In spite of whatever method used in determination and identification of the donor and
recipients all the way through the process, we as Medical Laboratory
Scientists/Technologists should be aware of the adverse consequences of mislabeled or
misidentified donors or recipient before and after blood collection. This is to track any
discrepancies or mistakes in collection process and crossly mislabeled and misidentified
patients that might happen through routine collection and testing, although all blood
donated will be screen for infectious diseases and we try to be as meticulous as
possible however the likelihood of misidentification and infection are always lure as a
danger to the recipient (Rodgers GP et al).
Some donors will be deferred for unlikeness of their contribution for the unsuitability of
their blood itself such a high blood pressures, some illness (malaria or HIV, hepatitis
and CMV. etc.) or presence of some chemicals (drugs etc.) in their blood. The deferral
may include track marks as sclerotic vein (this is a case of permanent deferral).
Guidelines of safety in transfusion medicine service have been forwarded by American
Association of Blood Bank (AABB), Department of Health and Human Services,
FDA (Food and Drug Administration), Canadian Blood Services (CBS), and others. A
thorough clinical history is the key to a safe acquisition of the donor’s blood. Deferral
is a list of conditions that rejects donor either permanently or temporarily. Donors
selection includes medical history, confidential unit exclusion (CUE/for illness that
need to be excluded without identifying the subject’s source or the donor with illnesses
such as AIDS, etc.) and physical examination (as weight, temperature, blood pressure,
pulse, PVC (Pack Cell Volume/hematocrit) or hemoglobin (Hgb.).
Collection consists of materials that must be used during the collection process and must
be sterilized all the way to protect the donors and recipients. Next to collection process
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
is the containers: these are special vessels that contain anticoagulants and
preservative to prevent coagulation and extent the shelf-life of the blood unit.
Subsequent to materials and containers we have identification as mentioned earlier and
the phlebotomy initially: ask the patients name and confirm the identification by three
identifiers as social insurance number, name and OHIP card (Provincial Health Insurance
Card) or valid ID.
Selection of the vein during phlebotomy is similar to blood collection for Clinical
Chemistry and Hematology lab work ups (refer to these parts (I and II); in this type of
blood collection the donor should squeeze hand every few seconds to facilitated the
flow of blood into the bag, the only exceptions are the needle gauge (#16), collection
containers set ups and bags as well as the needle assembly itself. In respect to collection
bags they consist of primary bag, the collection satellite bag number 1 and satellite bag
number 2 attached together by tubing. They are added with CPD (Citrate Phosphate
Dextrose/earlier version was ACD/Acid Citrate Dextrose) as anticoagulant and
preservatives. 2, 3 DPG (diphosphoglycerate) influences the release of oxygen to the
tissues (oxygen dissociation curve). Sodium bi-phosphate added to the unit maintain
the high PH during storage to maintain 2, 3-DPG [refer to clinical chemistry- part one
(1)]. Sometimes we add adenine to the CPD, making it CPD-1. In CPD-2 there are extra
dextrose molecules to this kind. Shelf life of red cell pack additive solution is 42 days
when stored at 1-6°C temperature (National Health and medical research
council/Australian…).
In respect to the sample collection, before venous access, once the vein has been selected
the iodophore compound such as povidone iodine or Betadine or so on, it may be
used to disinfect the area. Scrub for 30 seconds and for the second time it should be
disinfected in concentric circles while moving in an outward direction disinfect the
location, after the disinfection done scrupulously, pierce the vein and have the venous
access and collect according to the guidelines. Before collection the tubing must be
clamped with a hemostat between the primary bag and the needle as indicated, after the
insertion of the needle, it (the hemostat) should be release to collecting in the bag through
blood flow in the tubing.
During collection the collecting bag must be swirl to admix the preservatives and
anticoagulant properly so that the unit will last to its desired shelf-life. The pilot tube or
the specimen tube (vacutainer) attached to the main bag or separate may be filled
properly by inserting the needle after removal from the donor’s arm to the vacutainer’s
tube stopper, or to do “cut and drip” method which consists of removing the unit by
sealing the tube while applying a hemostat (to control the flow of blood into the tubing)
above the seal then cut the tubing bellow the hemostat and above the seal (while needle
is in the patient’s arm), this will allow flow into the open specimen tubes. Collecting
pilot tube and bag should be done before the donor’s leave the room at his bed side
(Quinley DE et al.).
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After the collection the donor must be cared for and kept in the bleeding center for at
least 30 minutes to observed of the adverse reaction of the phlebotomy by giving juice to
build the blood pressure and bring him/her back in case of fainting. In this section I will
explain the adverse reactions after phlebotomy and reaction grades (blood donation
reactions) at the end of this section, these reactions are different from the transfusion
related reactions, which are hemolytic and will be discussed in chapter four ”post
transfusion reactions”.
RH typing (Downes KA et al. & (AABB manual)
One of the major parts of blood banking is typing for blood groups such as Rh and
ABO/ABH or some other important blood grouping deviations such as Rh° (D) of Rh
blood group or e, c or weak- D antigens and ther subgroups of blood group systems.
Rh typing is done by testing blood with Rh antiserum (anti-D). The Patient’s or
recipient whole blood is typed by Rh antiserum. This is to confirm Rh antigen on the
recipient or the donor’s red blood cells. As for donor’s blood this would be tested with
these reagents to confirm donor’s and recipient’s Rh blood type which is needed further
for DAT, IAT, cross match and antibody screen determinations.
If the Rh antiserum agglutinates with the whole blood of the recipient, it will indicates
that blood Rh type is “Positive” for Rh antigen on the red blood cells, if there would be
no agglutination this means there is no Rh antigens present on the red cells. The test
before reporting as positive should be tested for weak D antigen (this will be explained in
the Rh blood system), if weak D agglutinates (due to antigen-antibody binding), the
blood type is Rh positive, while reverse is true when there is no agglutination with weak
D-antiserum, therefore the Rh type is negative. Usually routine testing for other blood
group type is not necessary and ABO/ABH and Rh grouping will suffice. The
following is example of a positive and negative (slide and rapid tube tests) Rh testing
with antiserum with or without agglutination (Fig, 1-1).
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(A)
(B)
Fig. 1-1: (A) a positive Rh°(D) Rh typing, and ABO typing slide test and (B) a positive
and negative Rh blood typing in rapid tube test (courtesy of Serafol®), note the clumping
or agglutination in two positive cells and the negative cells in the middle.
Rh typing can also be done in tube as a rapid tube test for confirmation of doubtful slide
test result. It may be done by placing a drop of 5% suspension washed whole blood in
the tube and equivalent amount of Rh antiserum (anti-D) in the tube and gently tilt tube
for agglutination: the slide testing consists of a drop of recipient whole blood on the slide
and one drop of Rh-antiserum will be added by applying and tilting on a 37°C heater
tile/block (Rh viewing box); a positive agglutination is the clumping of red cells, a
negative has no agglutination/clumping.
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ABO typing
ABO blood group includes testing for ABO antigens or antibodies in the either whole
blood or the serum of recipient or donors: the testing is the same procedure as Rh testing
with difference of antiserum and antigens specific for ABO antigen or antibodies
respectively. In this typing we either do slide test or rapid tube test; in slide test a drop
of whole blood or the serum of the recipient or donor is added to the slide and a drop of
antiserum or antigen in that order would be added to the slide next to the drop of whole
blood or serum then they are admixed and tilted gently to facilitated agglutination if
present. There must be no heating in this procedure. This is a forward grouping [or cell
typing, when we add ABO antiserum to the whole blood (red cells) to determine ABO
blood type]. In backward (reverse/serum typing) grouping/typing the antigen (as
cell reagents) commercially prepared will be added to the serum of the recipient or the
donor then would be mixed and gently tilted and the result would be registered. The same
as Rh typing in ABO typing the rapid tube testing is done by adding a drop of antiserum
of each blood group in each tube and adding 5% suspension of whole blood (or serum
in reverse grouping) to the tube and each reaction (of A, or B antigens) will be reported
as positive for clumping of cells (agglutination) and negative for the lack of it.
The figure 1-1 shows a positive “A antigen” and a negative “B antigen”, an O positive
blood group (universal donor type) is when there is no clumping in both A & B
antigens with the corresponding antisera, for AB positive (universal recipient type)
blood group there is clumping of both A & B antigens with corresponding antisera: as
said reverse is true when the blood group A and B are negative for antigens with the
correspondent antisera used (O type) A type and B type bloods follow similar process.
See the pattern of agglutination in the figure 1-1 (A): if the slide test is doubtful a rapid
tube test with 5% red cells suspension should follow. The result of forward grouping
must match with reverse grouping to confirm the results. 5% suspension of red cells
may be made by diluting whole blood by saline, serum or plasma and centrifuging it for
10 minutes for 5 times and decanting the supernatant and washing the infranant or the
RBC pellet between spinning (Downes KA et al/Rodgers GP et al).
Detection of unexpected antibodies to RBCs antigens (Quinley DE et al
and Natiional Health and Research Council/Austrilian…)
This is to detect antibodies to BRC (Red Blood Cell) antigens (due to
alloimmunization) and is done by means of antibody screen and identification. AABB
& CBS (American Association of Blood Bank and Canadian Blood Services) recommend
this be done on donors with history of transfusion or pregnancy. However it is easier
and can be performed on a routine bases for all blood units, under this heading we have
typical antibodies for ABO/ABH alloimmune antibodies and atypical antibodies
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such as antibodies to other blood groups systems allomminzation (Lewis, Kidd, Kell,
etc.). In addition, unexpected cold reactive autoimmune antibodies such as anti-I & anti-i
sensitization with this type of autoantibodies may be present in cases of autoimmunity
with IgM sensitization. For unexpected cold reactive antibodies refer to I blood group
system with IgM sensitization.
The unexpected antibodies are provoked in the recipient against RBC different blood
group systems of the donor during or after (alloimmunization) of a mismatched blood
unit infused into the recipient. As with this we have to use methods to screen and identify
these antibodies. Methods may include LISS/Coombs and absorption, elution
techniques using proteolytic enzymes (such as papain, ficin, and pepsin, trypsin and
chymotrypsin etc.) and chemical substances such as 2-ME (2-mercaptoethanol), PEG
(polyethylene glycol), DTT (dithiothereitol) and ZZAP (a combination of proteolytic
enzymes and thiol reagent as DTT).
In LISS (Low Ionic Saline Solution) this agent would be added to donor’s serum to
recipient red cell antigens to potentiate the uptake of donor’s antibodies to recipient’s
antigens in room temperature [IgM antibodies, (e.g. anti-I & anti-i etc.) rm. temp. 425°C] by reducing the ionic strength of reaction between the antigens and antibodies with
IgM antibody and thus reducing the incubation time of the reaction at room
temperature.
In the elution technique the use of weak organic acids, enzymes, chemicals and heat do
facilitate the separation (extraction/dissociation) of antigens/antibodies from the red cells
and by using the absorption technique (with the utility of some of these substances) the
antigens will be absorbed into the additional/reaction matrix such as rabbit red cells
stroma, patient’s own red cell stroma or latex particles. Example of these include 2-ME
which is a disulfhydryl molecule that disrupt the IgM molecules to different inactive
monomers in vitro thus freeing the cell antigen(s) of IgM antibodies to react with blood
group antisera for the purpose of identification of the type of antibodies in vivo.
Respectively, DTT is used for the same purpose as with 2-ME. Additionally, ZZAP
mostly utilizes for separation (dissociation) of IgG molecule from the red cell by
affecting (breaking up) the IgG monomer molecule to pieces [such as papain (into
three fragment/Fab2 & Fc) and pepsin (into two fragment/Fab & Fc)]. The thiol group
(sulfhydryl group of ZZAP will break up the disulfide bound bellow the first sulfide
bound (hinge region) of the IgG molecule into two fragment permitting the Fab
fragments of IgG molecule to open wider thus increasing the dissociation rate of IgG
molecule, this is used in antibody screening and identification of antibodies in absorption
technique, this as well will increase the rate of binding of IgG antibody to the relevant
antigens.
Also IAT and DAT (indirect and direct anti-globulin testing/Coomb’s test) will be
performed to differentiate between IAT which detects:
1. Unexpected antibodies detection and identification
2. Antibody titration
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3. Red cell elute testing for detection and identification, and
4. Compatibility testing
And DAT detects and is used for:
1. Autoimmune hemolytic anemia (AIHA)
2. HDN
3. Hemolytic transfusion reaction (HTR), and
4. Drug induced hemolytic anemia (DIHA)
These (IAT & DAT) increase the antibody (in the blood group antisera) binding to red
cells antigens reaction by a potentiating (as an extra-joining ( as a bridge) molecular antiIgG piece between antibodies and antigen) agents such as human antiglobulin
reagent (AHG/anti IgG): this may be added to the IAT reaction at 37°C and or to DAT
at room temperature. The 6% bovine albumin is used as control serum for these
reactions. The procedure for DAT is the same as for IAT where the only exception is that
there is no incubation (37°C) for DAT with different applications. Figure 1-2, shows the
human antiglobulin antiserum binding to antibodies to red cell antigen.
Fig. 1-2: a schematic representation of AHG test (anti-human-globulin test) as a bridge to
facilitate agglutination of antibodies (alloimmune antibodies) to red cell antigens (blood
groups).
Various types of blood donation
Traditionally blood was transfused from a homologous donor (from the same species/
humans) for homologous transfusion who has no relationship to the recipient except
the matched blood type with the recipient. However in spite of the new methods of
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donation, such as autologous donation and or directed donation, the homologous
transfusion still remain the safest way among the recipients and donors. In here I explain
briefly the homologous type donation (vide infra) and the directed donations, while
the autologous donation will be discussed under the autologous donations at chapter three
in part four of this text (Quinley DE et al).
As per se the homologous donation is between two different members of the same class
as different individuals. In this type of donation donor’s blood will be matched against
the recipient of the subjective blood as a conventional blood transfusion. However
directed donation is when the patient’s make request to be transfused with his/her closest
family member: in this type of donation, blood is not necessarily matched against the
close or immediate family member the idea behind this was a heightened awareness of
public concern/opinion about AIDS and HIV high prevalence rate during 1980s. This
awareness led to transfuse with their immediate family member’s blood at the time
necessity by some people. But directed transfusion has shown to be no safer than
homologous donation not to say is more disastrous due to high rate of GvHD reaction
(Graft vs. Host Reaction/Disease) which can be very fatal. In this type of donation the
donor’s blood will be irradiated to reduce the surface markers (human
histocompatibility/Human Leukocyte Antigens/HLA) so that to decrease or
prevent provocation of immune system response to foreign HLAs in these types of
reaction in the host or the recipient of the purposed blood.
On the contrary in autologous donations the person’s blood is collected/donated
sometime prior or during the operation/surgery and stored to be infused back to the
recipient at the time of necessity for these blood units. This type of donation
(autologous) will be discussed further at chapter three of part four.
Adverse reactions & Reaction grades at voluntary blood donation
(klein et al)
The donor reaction during phlebotomy is considered here as reaction grades this
consists of grade I, II and III reactions, in reaction grade I, the donor usually tolerate the
phlebotomy quite well however some may experience side effects and adverse reactions
such as grade reactions. The state of the mind or psychology of donor may play a role
as well in developing the post-phlebotomy reactions. In this regards, nervous donors
experience more grade reactions. In grade one (1) reaction he/she may show symptoms of
paleness, weakness, hypotension, dizziness, nausea, sweating, rapid pulse, rapid
breathing, twitching/muscle spasm and may occasionally faint (syncope), these could be
due to nervousness or due to unknown cause. If the grade I symptoms occur during
phlebotomy immediately remove the needle and the tourniquet from the arm and
explain to the patient to breath slowly and deliberately/deeply.
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In grade II reactions the symptoms, donor may be nauseated, puke or vomit for these
reason the head must be place side way to prevent aspiration of the vomitus into the
pulmonary sacs and follow the prerequisite first aid measures and guidelines to
counteract the symptoms: a cold compression must be applied to the head and the back
of the neck, explain to the patient to breath slowly and deliberately/deeply. Vital signs
(as blood pressure, pulse and respiratory rate) must be monitored and donor’s legs must
be elevated higher than the heart and the brain. Release the neck tie or loose the collar.
If necessary use ammonia inhaler and allow him/her to breathe in the paper bag and cover
the donor with blanket to prevent heat loss.
In type III reactions or systemic reactions which consists of all previous grades including
vasovagal in nature, involuntary muscle spasm and contraction, cyanotic color,
suspended respiration, dilated or fixed pupils and urinary and fecal incontinence
(gastrointestinal disorder/loss of sphincter control), with muscle spasm and convulsive
syncope and tetany plus the signs of circulatory (or cardiovascular) shock and
bradycardia, and there have been reports of rare cases of ACD intoxication causing
severe arrhythmia, the scientist or the technician must stop the phlebotomy by
cautiously removing the needle from the arm to prevent further injury to the donor and
preform all the above first aid treatment. The airway must be kept open with
adequate oxygen. Care must be exercise to prevent the donor from falling off and out of
the chair. The evaluation must be done to see whether the donor is acceptable to donate
blood by future time or not.
Chapter Two (2)
Definition and types of Hemapheresis (Rodgers GP et al/Quinley et al)
S
eparating devices are used in the preparation of blood components and their
products. These use centrifugal force to separate components. These
technologies include centrifugal methods; bowl technology; Continues Flow
Centrifugation systems (CFC); separation by membrane filters and separation by
adsorption. The hemaphreresis (apheresis) are called to when different components of
blood (such as platelet, pack red cells and granulocytes, FFPs (Fresh Frozen Plasma) and
cryoprecipitate, and some coagulation factors or stem cells) are separated by these
technology, these are used in extracorporeal therapy. The schematic presentation of
bowl system and CFC machine has been depicted in the following picture just as a model
to show its infrastructure (Fig. 2-1), each procedure has its own advantages and
disadvantages. In this systems of apheresis the blood is collected from the vein and
separated into the desired components and then would return to the donor’s circulation in
a continues bases or cyclic (intermittent) forms from the portal of exit to the
device/machine and back to the patient’s vein again to his/her blood stream through
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portal of entry into circulation. As mentioned, apheresis is the separation of all
components one at each step by these separation systems.
Other technologies are used with similar application such as intermittent flow
centrifugation: in this system the flow of blood to the device is cyclic and
intermittent.
(A)
(B)
Fig. 2-1: (A) the design of apheresis unit for bowl system, and (B) the actual infrastructure of
apheresis unit for blood components, the system can separate platelets, leukocytes and red cells
either from buffy coat or plasma respectively, the stem cells may be separated with the same
technique and device.
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Extracorporeal circulation consists of attachment of the circulatory ports (such as cannula,
fistula) to the apheresis unit for the purpose of separation of the blood components: the term also
applies to the lung-heart-machine and extracorporeal oxygenator.
As said, apheresis and hematopheresis means to separate whole blood to its components and
final products. The thrombocytopheresis is the separation of thrombocytes/platelets from
buffy coat layer (part two, hematology), erythrocytopheresis is the same process of separation
of blood red cells (erythrocytes) from the whole blood by removing plasma, and buffy coats. The
lymphocytopheresis is the separation of lymphocytes from the circulation by apheresis
technique from the buffy coat layer, and the leukocytopheresis is the separation of
leukocytes or granulocytes (granulocytopheresis) from the whole blood or particularly from
buffy coat. The lymphocytopheresis is needed for the patients who suffer from cell mediated
immune deficiency, while eryhtrocytopheresis is used for patients with tissue oxygenation
demand/requirement. In addition to the above, the thrombocytopheresis will be given to
patients in need of platelets who suffer from thrombocytopenia, this product must not be given to
TTP and HUS (Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic
Syndrome), where it can cause fatal derangement of the coagulation system and platelets
destruction in the host and worsening condition of the patient rather than helping to recover.
Leukocytopheresis is utilized for patient with possible infections and leukocytopenia, this
should weight against patient’s signs and symptoms to prevent against possible GvHR reaction
(graft vs. host reaction) which can be lethal. The same precaution should be exercised with
lymphocytopheresis administration to immuno-competent host which adversely may cause
GvHR reaction and invariably might be fatal, it may be used for immunosuppression, immunecompromised patients: it should consider with caution due to HLA antigen on the leukocytes and
lymphocytes with either anamnestic response or immediate GvHD (graft vs. host disease), The
leukocytopheresis and lymphocytopheresis may be irradiated before administration to reduce the
morbidity or may even mortality of the patient.
Blood Components (Downes KA et al)
Whole Blood
Whole blood is the extraction of the blood itself as with the anticoagulant and preservative
mentioned previously without any further separation or fractionation of the components or the
whole blood itself.
The amount of the package (unit) is 450 mL with a 63 mL of approved anticoagulant. The unit
must be kept at 1-6°C for about 21-35 days for storage purposes. The hematocrit should be
between 35-40% (0.35-0.40), and the red blood cell mass contains 180-225 mL. We have to
consider further that the labile factors such as coagulation factors V and VIII along with
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leukocytes and platelets functions are ineffective in whole blood unit due to suboptimal
condition of the whole blood storage.
As in modified whole blood, the plasma (70 mL) and platelets along with cryoprecipitate has
been removed by the previously mentioned methods although other criteria of whole blood unit
are the same for modified whole blood with whole blood.
Red cell concentrate and random leukoreduced concentrate
(Quinley et
al/Rodgers GP et al)
This component has been fractionated by centrifugation from whole blood. The unit has its
platelet (usually removed during process) and leukocyte removed accordingly and in short term
storage these (platelets) if not removed become nonviable (1-6°C). The RBC mass is the same
as the original whole blood mass and they should be kept at 1-6°C for 21-42 days storage in the
fridge with requisite temperature for liquid stored RBCs. For this the amount of anticoagulant
and the preservative must be considered in setting up the storage. The leukocytes-reduced
concentrate units can be prepared from this unit. The unit of red cell concentrate may be
washed frozen-thawed and deglycerolize (high glycerol is used to freeze the red cells), so that
to prepare leukoreduced red cell concentrate by this unit with this method.
Indications for above components is related to conditions such as anemia and low tissue
oxygenation, this is for to increase oxygen delivery to the host tissues and reduced the effects
of anemia. This may be indicated by increase red cell mass. There is no standard level of
hemoglobin or hematocrit to which the infusion would be based. The unit can be used for
normovolemic patient with anemia. Patient with anemia with signs (symptomatic) of anemia
without active bleeding may be benefited from these products as these patients can tolerate
excess hemoglobin up to 70 g/L (Hct. 21%). The need for higher doses of red cells concentrates
and whole blood with higher hemoglobin level indicated in Coronary Artery Disease (CAD)
and cerebrovascular insufficiency. Usually patients can tolerate 20-25 % blood loss during a
hemorrhagic episode without any need for blood replacement, in this degree of loss heart rate
and peripheral vascular resistance will increase to replace and cope with the loss of blood,
the fluid shift will concomitantly occur from extravascular compartments to intravascular
space to maintain the volume, blood pressure and the number of red cells circulation in the blood
stream. In this situation the volume expanders and intravenous fluid may be used to increase
blood and tissue oxygenation.
Counter indication of these products/components is when the unit will be used for volume
replacement and as nutritive source. In this approach it is not recommended to utilize whole red
cell or red cell concentrates for intravascular volume replacement. Instead of this option for
volume replacement the volume expanders may be used for blood volume augmentation such
as albumin/colloids, etc. The whole blood or concentrate must not be used as a tonic as well
as for nutritive purposes and has no indications. Patients with infection and concurrently
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anemic should not be treated with these components as these have not been documented or
reported by any sources.
Random platelet concentrates and platelet concentrates (BCSH et al/ German
medical society/American society of clinical oncology & Italian society of transfusion medicine
and immnuohematology)
This random concentrate has been concentrated from the buffy coat layer fraction of the whole
blood by the thrombocytopheresis. This apheresis unit uses for the platelet transfusion
therapy and should be administered to patients with thrombocytopenia. The unit contains at
least 55 x 10 10 platelets in the unit with platelet suspension into a 50-70 mL of plasma. This will
be stored at 20-24°C for 5 days with consistent agitation. In platelet concentrate (contrary to
random platelet concentrates) or platelet apheresis the storage requirements is the same as
random platelets concentrate cited above. Random concentrates at least must contain 30 x 10
10
platelets suspended in a 200-400 mL of plasma in each unit. Storage period is 20-24°C for 5
days with agitation approved by FDA (food and drug administration). If leukocyte were not
removed during preliminary separation, it may remain as contaminants in this unit.
Indications are of prophylactic platelet therapy in non-immune thrombocytopenia with
platelet count below 5 x 10 9 (below this level hemorrhage is evident) except when the patient is
experiencing long term non-immune thrombocytopenia or is refractory- when there is no
platelet response to the thrombocytopenia. And indications may be applied also when platelets
are less than <20-30 x 10 9 in patient with other hemostatic complications such as liver
diseases with impaired coagulation, DIC (disseminated intravascular coagulation), sepsis,
anticoagulant therapy, fever, necrotic tumors and antithymocytes therapy etc. Other
indications when platelet count is below < 50 x 10 9 in patients scheduled for lumber puncture, or
platelet transfusion in patients with hemorrhage and platelet function defects or
thrombocytopenia, in patients with autoimmune thrombocytopenia (this should be weighted
if appropriate). As well platelet transfusion would warrant in massive hemorrhage, and
platelet congenital disorders and children under 4 months old.
Contraindications of prophylactic platelet therapy are in Thrombotic Thrombocytopenia
Purpura (TTP) whereas it makes the situation complicated, or in Heparin Induced
Thrombocytopenia (HIT syndrome), also contraindicated in congenital IgA immune-deficiency
as it may cause anaphylactic shock in these patients. Platelet transfusion is not indicated as
well in ITP (Idiopathic Thrombocytopenic Purpura) or post transfusion purpura- in these,
the patient may be prone or at risk to infection- and this therapy (platelet) is not warranted in
NAIT (only infant’s mother platelet concentrate may be used/NAIT-Neonatal Alloimmune
Thrombocytopenia).
Granulocytes component (Downes KA et al and McVay PA et al)
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
The component is derived by granulopheresis concentrates (apheresis of granulocytes), it is
prepared from buffy coat of fresh whole blood. Granulocytes apheresis unit may contain 1 x
10
10 granulocytes. RBCs and platelets may be collected during this process and because the
amount of RBC are present (20-50 mL) it is essential to do an ABO group cross matched with
the recipient. There is 200-300 mL of plasma along with the anticoagulant in this unit.
Granulocytes are indicated in bacterial and fungal infections with severe neutropenia with
leukocytes less than 0.5 x 10 9 . Patients with congenital neutrophil defects (chronic
granulomatous disease) can benefit from granulocyte concentrates. There have been reports and
documentation that patients with refractory or resistant bacterial and fungal infection to
antibacterial doses may be candidates for granulocytes apheresis concentrates if they suffer
from reversible myeloid hypoplasia (bone marrow aplasia). The granulocytes concentrates also
has been used in granulocytopenic and thrombocytopenic patients with bone marrow failure
or under chemotherapy.
It is indicated in situation such as infections uncontrolled by standard and conventional
medical therapy and is contraindicated in most instances of marrow transplantation therapy
where it has been shown ineffective with the low rate of survival of the patients with these
conditions. This is a transfusion granulocytes adjuvant therapy and must be given: it
should be given only when there are no other conventional medical treatments available.
Prophylactic granulocytes transfusion is not justified in all septic and other treatment cases for
the following live threatening situations that might follow as in:
 Hemolytic Transfusion Reactions (Acute and Chronic HTR)







Febrile non-hemolytic reactions
Allergic reactions ranging from urticarial to anaphylaxis
Septic reactions
Transfusion Related Acute Lung Injury (TRALI)
Circulatory overload
Transfusion associated GvHD (Graft vs. Host Disease)
Post transfusion purpura.
Granulocytes possessing HLA marker will provoke GvHD and can be fatal, it will cause the
above adverse reactions if given intravenously (IV) and should be pondered and weighs its
disadvantages against the advantages.
Fresh frozen plasma (FFP)/(National Health and medical research council…Australia/
& McVay PA et al)
This products/component is obtained from whole blood by separating and freezing the plasma
within 8 hours of attainment/collection, one unit consists of 250 mL liquid plasma. These once
prepared for transfusion it must be matched with the recipient’s RBCs ABO blood group. It must
be kept in temperature (storage) at about -18°C or lower for a year. The components contain all
factors and elements of blood plasma such as fibrinolytic proteins, immunoglobulins,
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
isohemoagglutinin antibodies, fibrinogen, complement components, coagulation factors, oncotic
proteins and other relevant proteins and elements in the plasma such as alpha, beta and gamma
globulins. Because of the presence of isohemoagglutinins in this fraction it must be ABO
group cross matched and thawed before transfusion in 37°C water-bath and can be used up to 5
days after thawing It is free from red cells, leukocytes and platelets because there is no
leukocytes therefore there will be no risk of CMV (cytomegalovirus) transmission. The
following Plasma ABO typing for infusion to the related blood type must include: unknown
patient’s antigen will be given plasma AB (if required urgently); for O type patients, the plasma
transfusion must be A, B, or AB or O. For patients with A type blood, plasma transfusion must
be type A, or AB; As with patients with B type blood, plasma transfusion must be B or AB and
lastly for patients with blood group AB, plasma transfusion must be type AB (plasma should be
type A for transfusion if AB unobtainable). Plasma should be thawed in 37°C water bath before
transfusion.
Solvent-detergent treatment of FFP has been used to prevent infectious disease transmission
such as HIV, hepatitis, etc. however its advantages and safety must be balanced against its
disadvantages and cost of production. Solvent-detergent plasma has all the indications,
contraindications and criteria to FFP. However as mentioned it should be given with
consideration to the cost of production.
It is indicated in active bleedings, coagulation deficiency, which could be acquired secondary
to liver diseases or congenital or for people who are having invasive surgeries.
The deficiency of factor IX as in Hemophilia B should be only transfused when the factor IX
concentrates are not available or in the absence of recombinant or factor VIII concentrates in
Hemophilia A. The deficiency of other factor has indications for FFP plasma therapy.
Coagulation deficiency may be in form of warfarin anticoagulation, DIC (or Disseminated
Intravascular Coagulation), liver diseases, or massive replacement of red cells or crystalloids
and colloids. Vitamin K can be used for patients on warfarin anticoagulation so that to
recover the patient from warfarin effects (reversing the action of warfarin). Patients with massive
transfusion of whole blood can be benefited from administration of FFP for these patients are
usually thrombocytopenic therefore is beneficial: transfusion of platelets concentrates would be a
better choice in management of the patient. Prophylactic FFP therapy is also useful in
immunodeficient patients (only when there is no IV-intravenous- immunoglobulin available)
although a better choice is the intravascular (intravenous) immunoglobulin therapy. And the
last but not the least it can be indicated in TTP through plasma exchange.
Contraindications (adverse effects) include when the physician cannot correct the problem
with conventional and standard medical interventions by using vitamin K replacement,
recombinant or factor VIII & IX concentrates or by cryoprecipitate, or other factor specific
concentrates, therefore it may be used. Also must not be used in plasma exchange procedure
except in TTP (Thrombotic Thrombocytopenic Purpura), it should not be used as a
treatment of immunodeficiency states as well (where it can be done by immunoglobulin
injections/IM or IV) and lastly in situation where treating hypovolemia due to hemolysis or
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
bleeding, in these cases plasma expanders such as 9% sodium chloride, crystalloids or
colloids are effective and the best choice.
Cryoprecipitate anti-hemophilic factor
This fraction of plasma which is cold-insoluble are obtained when fresh frozen plasma are
thawed at 1-6°C. It is white gelatinous precipitate called cryoprecipitates in FFP unit: it is
thawed slowly at 4°C to precipitate out. It contains plasma coagulation proteins/factors
including 80 units of VIII:C factor; 250 mg fibrinogen; 20-30% factor XIII and 40-70% of
VWF (von Willebrand factor): it contains about 20 mL in volume. It must be stored for one year
at -18°C as storage time and temperature. It should be used as needed in situation where other
conventional and standard therapeutic approach is unavailable. It is used for the above factor
deficiencies and as a primary indication of hypofibrinogenemia it may as well be used in the
diathesis of uremia unresponsive to DDAVP (desmopressin acetate vasopressin). It is also
used in treatment of VWF only when there is no VWF (Vvon Willebrand Factor) plasma derived
factor of VWF available or DDAVP is ineffective (contraindicated). Vessel patents and local
sealant or in otic surgery are suggestive and used when cryoprecipitate mixed with thrombin to
produce “fibrin glue”. The volume of infusion must be considered not to exceed or approaches
the blood volume as it will cause hemolysis of the ABO matched RBCs.
Clotting Factor Concentrates (Quinley et al/Klein et al))
The clotting factor concentrates usually are prepared by fractionation of plasma pool with
immune-affinity column chromatography purification, precipitation, ion exchange
chromatography and or by recombinant technology. These concentrates are usually is for
replacement of factor VIII for hemophilia A or acquired factor VIII inhibitors and vWF
deficiency if indicated by the manufacturer and factor IX concentrates for hemophilia B (or
Christmas disease). The concentrates are used instead of FFPs and cryoprecipitate as the
supplement for these factor deficiencies. This is for the purpose of reducing the substantial
risk of transmission to the recipient of infectious diseases or AIDS/HIV.
Other components may include immunoglobulin shots and numerous other components such
as colloids as plasma proteins and albumin (and crystalloids as dextrose) for intravascular
volume replacement. It controls blood pressures and used for patient going into shock or
surgeries. IV (intravascular) immunoglobulin preparations are utilized mostly in
immunodeficiency syndrome and in immune-compromised host with immunoglobulin
deficiencies In cases of platelet refractories in platelet concentrates infusion where HLA,-A & B
on platelet might provoke immune response to platelets and therefore reduce their survival and
its efficacy, immunoglobulins preparations may be given to coat the sites on the reticluoendothelial systems to prevent stimulation of immune response to platelet acquired HLA- A
& B antigens and therefore better survival of platelets. For platelet reconstitution and infusion
recombinant thrombopietin is available for RBCs the recombinant erythropietin constituent are
Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
534
produced by manufacturers of diagnostic reagents by genetic engineering methods. And lastly
the growth factors such as Colony Stimulating Factor (e.g. CSFs) to fortify bone marrow
components may be used. These CSFs include G-CSF (Colony Stimulating Factor –
Granulocytes) for white cell and GM-CSF (or commonly known as Granulocytes MacrophagesColony Stimulating Factor). All the said factors are used in cancer therapy, infection with
depleting granulocytes/leukocytes and anemia, for transfusion purpose especially thrombopietin
and erythropietin may be used to increase tissue oxygenation and hemoglobin/erythrocytes
concentration to counterbalance the blood loss.
Blood Group Systems (Reid ME et al)
ABO blood group system
The ABO blood group system was discovered by Karl Landsteiner in 1900 & 1901 and was
given Nobel Prize for his discovery of ABO blood group system in 1930. The ABO system
consists of ABO types and there are only two antigens and two antibodies responsible for
outcome of the ABO blood types. The blood typing in this system includes A, B, AB, and O
types. These two antibodies are anti-ABO isohemoagglutinins/isoagglutinins and are
responsible for transfusion reactions as in here with ABO blood groups: isoagglutinins are
antibodies that can be produced by alloimmunization with ABO and other blood grouping
antigens and systems with correspondent antibodies. The monoclonal technology has given way
to synthetically producing these antibodies in form of grouping antisera and antigens in vivo.
This system of antigen and antibodies (or better to say, blood group system) is the most
important blood groups in comparison with other blood group systems. Incompatibility of these
blood groups may cause severe transfusion reactions to next potent antigens of Rh°(D)
antigens (Rh system). A combination of these ABO types determines the ABO blood groups of
the individuals. The antigens are present in the blood type of individuals as positive for that
antigen it means the blood type is positive. Example is A antigen positive would be blood group
A and or B antigen positive would be B blood group individual: the rest of the ABO grouping
would be the same: refer to the table 2-1 indicates different blood group types with presence (+)
or absence (–) of the antigen or antibody.
Table 2-1: The ABO antigen and antibodies in different ABO blood
groups
Blood
groups
types
A
B
O
AB
Antigen
Antigen Anti-A Anti-B
A
+
–
–
+
B
–
+
–
+
–
+
+
–
+
–
+
–
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
–= not present, += present
These antibodies before were known as naturally occurring isoagglutinins however now it is
called non-red cell stimulated antibodies (NRCS) for the reason that they have proven to
occur during neonatal periods when the neonates are exposed to correspondent A or B antigens
in the environment before 6 months of age. The alloantibodies are antibodies provoked
(alloimmunized) to another member of the species red cell antigens (other human being with
different antigens) by exposure to the donor, pregnancy, or transfusion of a wrong ABO blood
group antigen typing or erroneous cross match. Example is when an A type individual exposes to
a blood products (through transfusion etc.) to blood group B types individuals in this scenario the
antibody (Anti-A) A in the donor erroneously transfused (blood typing errors) to the recipient
will reacts or combine to antigen A on the recipient’s red cells agglutinating them and
resulting in hemolysis of some cases. This is the same for other ABO blood group types.
Individual with group O blood type do not have both (A, B) antigens on their red cells and thus
will not react with other A, B, or AB types as these type blood and group of people are called
“universal (Blood) donors”, but they can receive blood only from type O individual for they
both antibodies anti-A, and anti-B can react with all other ABO blood groups, recipient
antibosies (O type) will have much less hemolytic reaction to AB type donors.
The reverse is also true with blood group AB individuals who have A, and B antigen and react
with all ABO blood group individuals, and that are why they are “universal (blood)
recipient”. They can receive from group A, group B, and AB and O because all are positive for
A & B antigens, but however the AB group have no antibodies so it reacts with all other groups
(A & B, O) antibodies that have no A or B antigens on their red cells and they are positive for the
corresponding antigen(s), as A, B, and O, therefore they cannot donate blood except to another
AB blood types. In spitte of this only much blood should be given to the patient.
ABO blood groups are inherited through alleles (the genotypes of the individuals) located on
chromosome 9 and are manifested through blood groups on the surface of the red cells or other
tissues (phenotypes). This pattern of inheritance does not change throughout the ontogenetic
development of individual by environmental changes or etc. Table 2-2 shows the alleles
(genotypes and phenotypes of the ABO blood group system.
Table 2-2: Genotypes and phenotypes of ABO blood group system
Parents
allele
A
A
B
O
AA
AB
AO
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
B
(A)
AB
(AB)
BB
(A)
BO
O
(AB)
AO
(B)
BO
(B)
OO
(A)
(B)
(O)
(A), (B), (O) & (AB)= phenotypes
AA, AB, BB, AO, BO, OO= genotypes
A, B, O= parents allele
The dominant characters in the punnett square, like A and B have phenotypes such as
dominant A and B from the genotypes AO and BO. Other character such as A and B are
inherited in a codominant fashion characterized by AB phenotypes: one A from one
parent and B from the other parent. It involves the occurrence of two genotypes (AB) at the same
time on the red cells. Recessive character such as OO shows as recessive O that has been
inherited from both parents. A and B phenotypes are dominant over O type thus an AO
genotypes will result to an A phenotypes etc. and for BO would be B. Agglutination pattern
of the antigens (red cells) and antibodies (antisera) has proved and documented this. The ABO
blood group typing has mentioned in ABO typing in chapter one of this part 4 refer to the section
for ABO forward and reverse (backward) typing/grouping (Reid ME et al and Quinley et al).
Bombay phenotype and ABH system
ABO antigens are derived from a precursor substance known as H antigen. The H locus is on
chromosome 9 and encodes a fucosyltransferase a substance that produces the H antigen on
RBCs. The H locus has three allele A, B and O: the A allele encodes an enzymes known as
“glycoltransferase” so that it will add “N-acetylglactosamin”to the H antigen’s D-glactose end
terminal on the red cells producing A antigen (type). For B allele, the induction of the enzyme
glycosyltransferase to add D-galactose to D-galactose end terminal of the H-substance on the
red cells, thus making it a B antigen. In respect to the O allele it lacks these enzymes and
therefore it cannot add these substances to the H-antigen consequently H-antigen remains silent
(amorphic or silent gene Hh/single dose) or unchanged. There are a number of people that lacks
H antigen activity (called hh phenotypes) which is known as “Bombay phenotype”. This
phenotype is quite rare (only 100 people possess it) and originally was found in Bombay, India.
H gene is present in single or double dose in all normal O type subjects/people and is necessary
for developments of ABO groups however, Bombay types is due to lack of production of Hantigen in terms of presence of hh gene (amorphic or silent gene/double dose) from each parents
which may happen in consanguineous marriages. Because Bombay phenotypes produce Hantibodies (due to lack of double Hh and presence of hh) to O phenotype people it
agglutinates types O. This is because of presence of Hh antigen in O phenotypes. In these
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
circumstances where O phenotypes agglutinates with another O positive, we usually type the
discrepancies (the Bombay phenotype) with plant products called” Ulex europaeus” or with O
known phenotypes such as Hh genotypes (O type), giving positive with the O cells possessing hh
phenotypes (Bombay). Other ABO antigens exist that has less effect on transfusion and is out of
the scope of this text. The subgroups of A and B antigens includes A 1 and A 2 with B 3 , B x and
or B m . For differentiation between A 1 and A 2 may be done with the plant extract “Dolichos
biflorus”. (More topics on this is in antibodies to ABO section below) (Quinley et al)
Antibodies to ABO
The ABO antibodies are divided into A-antibodies, A 1 -antibodies, anti-H and anti-A, B.
Starting with anti- A antibodies; this is actually in the sera of group B people with exposure to
antigen in the surrounding environment (NRCS/Non-Red cell Stimulating). This antibody
agglutinates group A and AB individuals. Anti-A agglutinates red cells in saline (saline
agglutinins) as these are IgM in nature (there may be a little amount of IgA or IgG present).
Accordingly the IgM antibodies (saline agglutinin) therefore can agglutinate red cells and
activates complement easily and cause rapid intravascular hemolysis of red cells carrying A
antigen. With this respect, anti-A may be divided into anti-A 1 1 which reacts with A 1 cells and
not with A 2 cells and anti-A common (may be a form of anti-A) which reacts with A 1 and A 2
antigens. In practice, initially the newborns who are actually A 1 type might be typed and
grouped as A 2 . This is because A 2 cells can remove anti-A 1 antibody by thorough adsorption
to the red cells, this is explained on the bases of presence of a common A antigen on the A 2 cells
(A common antigen). The A 2 cells has several branched epitopes of A cells (A antigen) present on
the surface while A 1 cells have fewer epitopes of A-antigen admixed with many A 1 antigens on
their surface. This is why as mentioned earlier neonates initially types as A 2 while actually they
are A 1 . This is all depends on the availability of the sporadic distribution of A antigen of the
A 2 cells on A 1
cells. The anti-A common antibody is a form of anti-A antibody as it reacts
differently with super-branched A 1 antigen (with several A antigen branches of A 2 cells) in
A 1 cells. Figure 2-2 shows the A 1 and A 2 cells with A antigen distribution on both cells. Plant
lectin D. biflorus once diluted can differentiates between A 1 and A 2 cells and use in
conjunction with anti-A 1 (adsorbed) and anti-A common for this purpose (Reid et al and Quinley et
al).
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Fig. 2-2: indicates the presence of few internal A antigens on the A 2 cells (quantitative
differences) may not provoke any antibody response to these cells as in typing of neonate blood.
Anti-H antibody is a cold reactive antibody (IgM) and can be found in A 1 and A 1 B
phenotypes and also on Bombay phenotypes (O). For more information refer to the above
discussion (vide supra). Anti A,B antibodies are present in O blood type individuals and can be
used to test weak subgroups of A and weak B antigens on A and B individual phenotypes or
for typing neonates with ambiguous phenotypes or confirming blood group O donors. This is not
simply a mixture of A and B antigen. The antibody (anti A,B) can react not only with A and B
cells but also has higher titer (affinity) and avidity (the strength of binding) than NRCS anti-A
and anti-B for A and B antigens.
ABO discrepancies
Sometimes discrepancies occur in ABO typing this consists of technical problem due to the
technologist errors and lack of SOP (standard operation procedures) or can be by other
means such as discrepancies in forward (cell grouping) and reverse grouping (serum grouping) ,
or with presence of interfering autoantibodies in the patient’s serum causing rouleaux
formation (this must not be confused with true agglutination: use microscopes to verify this/
rouleaux forms like the stack of coins on top of each other) or missing or weak antigens or
antibodies. The discrepancies should be resolved before releasing the reporting of the result to
the ward or the physician. In ABO discrepancies forward grouping might not match with
reverse grouping: i.e forward grouping shows A antigen while reverse grouping may show weak
or strong reacting anti-A (antibodies) with red cells. Therefore discrepancy exists between these
two; either anti-A must be anti- B or A-antigen must be B antigen to correlate with the outcome
of grouping. In autoantibody the autoantibody may cover the red cells and block the true
reaction or these could be other technical incompetence such as erroneous SOP or antiserum
expiration. Table 2-3 shows a case of resolving a discrepancy of Bombay phenotypes in ABO
grouping.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Table 2-3: Discrepancy in ABO grouping: a case of Bombay phenotype
The following table (2-4) also indicates a discrepancy between 2 twins with ABO discrepancy
the table shows the initial grouping and the follow up grouping and establishing an AB blood
type for the twin number 1 in comparison with both mother and the father of the twins in the
next table.
Table: 2-4: Initial group of the case study of a twins
If the autocontrol (O-type screening cells) in the ABO discrepancy be positive then an
adsorption and elution with rabbit cell stroma or patient’s own red cells may be used
to resolve the problem, if the discrepancy is in the forward grouping with antisera (cell
grouping) or vice versa is with cells (in serum grouping) either O cells (screening
cells) may be used for serum grouping: we may use heat (either increased or reduced
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
temperature-37°C or 4°C) to resolve the grouping in this way by using different
approaches. We can reach to a solid conclusion of a correct ABO grouping. If the
problem is the rouleaux formation and the typing is hindered due to formed rouleaux,
we can wash the cells with warm saline and repeat the screening. In cases of cold reactive
antibody/or autoantibody (panagglutinins: autoanti-I/in cold reactive antibodies use O
type screening cells) present in the serum and hindering the antibody (cell grouping)
phase, we may do adsorption with rabbit red cells and wash patient’s red cells with
warm saline to digress from cold antibodies (37°C): ABO incompatibility accounts for
major causes of HDN (hemolytic disease of the newborn) and other incompatibilities in
adults and neonates. It can be a cause of HDN in utero with mother being group O and
the fetus group AB
At last I define ploy-agglutination by “accidental exposure of some concealed antigens on
the person’s red cells surface to the individual’s immune system therefore producing
poly-agglutination of red cells”, examples include T activation and Tn activation: for
more trouble shooting/resolution of the discrepancies and ploy-agglutination concepts
refer to immunohematology text books or other transfusion medicine text books
mentioned/cited in the references.
Lastly the ABH system is Se (secretor) gene although secreted separately controls the
ABH antigens. A heterozygous Sese and homozygous SeSe or sese control the ABH
expression in such a way that H would be present. There is also a relationship between
ABH, SE and Lewis antigens (vide infra) refer to Lewis antigens.
Rh blood group systems (Daniels G et al)
The Rh blood group was discovered by Levine and Stetson in 1940. This blood group is
the second most important blood group system to ABO in causation of HDN (Hemolytic
Disease of Newborn) and other in utero sensitization and incompatibilities. There are
different systems of Rh nomenclature such as Fisher-Race terminology, Weiner and or
Rosenfield terminology however I explain them in brief so that familiarity with these
various types of terminology will not be totally lost, although the substantial information
is not the sole purpose of the part three in this text. Rh system name conveys from
rhesus monkey, which was the first animal experimentation that was done on this
animals by the investigators. The system may be Rh°(D) positive or Rh°(D) negative.
For the purpose of reporting the ABO blood groups and Rh blood group a conventional
manner exist. Example include if we have a positive B blood with a positive Rh type we
may report the patient’s blood type as B  or if negative Rh we will report as B  . This is
consistent with other positive ABO blood groups/types. In Fisher and Race system of
nomenclature we use D, C, E, c, e and G nomenclature or terminology whereas in
Weiner the conventional use of symbols such as Rh o , rh', rh", hr', hr" and hr G are
elaborated. The Rosenfield consists of Rh1, Rh2, Rh3, Rh4, Rh5 and Rh12, which has
less application. Table 2-5 shows comparison between Weiner and Fisher-Race
terminology.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Table2-5: Comparison
Terminology
between
Fisher-Race
and
Weiner
In brief in Fisher and Race terminology there are three closely linked set of
gene/alleles which are inherited with little or no crossing over between loci, in this d is
silent which practically does not exist, whereas in Weiner the product of a single
gene/allele at a single locus are the elements of the Rh system. In this system of
terminology each gene codes for an” agglutinogen” which conversely consists of
multiple blood factors. In this terminology agglutinogen is actually a haplotype and
blood factor(s) is Rh antigen(s). Each system has its own genotypes and phenotypes
the following table (2-6) is Rh haplotypes of Fisher-Race and Weiner terminology with
the relevant frequency in certain population.
Table 2-6: Rh haplotypes of Fisher-Race and Weiner terminology
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
The higher frequencies shows high occurrence of that haplotype while the how frequencies
indicated a lower occurrence in this definitive population (English islets). The different types
of haplotypes can be seen in the above table: example is CDe with 40.8 % and Cde with much
lower rate (percentages). Both Fisher and Race with Weiner has clinical applications while
Rosenfield has a lower applicability. The lower case characters in Fisher-Race system are
inactive or absent of the corresponding allele(s) whereas the capital or upper case letters in the
said system indicates active gene or presence of the allele(s) (Daniels G et al).
The five major Rh system yields (vide infra) the most probable phenotypes in Weiner. The
genotypes frequency can be used in determination of HDN in the certain population when the
frequencies are known and racial differences also play a major role in gene frequencies. Gene
(genotypes) frequencies, in such diverse populations as black populations, Asians, Hispanic
population, Caucasians are important to migration pattern and population genetic consensus as
well. Example is when a person has a haplotype of cDe (phenotype) in which the anti-c and
anti-e are negative however the anti-D is positive. Its genotypes may be frequently seen in white
population as cDe/cde (or R o r") where it would have much higher frequency in Black American
population and occurs as genotype R o R o .
The Weiner’s Rh system is important according to the antigens strength as the G, c, E, C and e
are significant in hemolytic disease: this strength is in decreasing order. To detect anti-D or
D-antigen there are recombinant antisera with IgG (it has an IgM or IgA class properties in some
low percentage), the IgG class can be potentiated by addition of albumin or high proteins or
other macromolecules to help increase the agglutination between them: IgGs. Care must be
taken to add and admix the antigen or the cells to cells or antibody respectively, there should be a
Rh control be run by the test to confirm the validity of the test.
D-mosaic property of the D-antigens and weakened expression of D antigens can effects
the outcome of sensitization with D antigens. (G, c, E, C and e), if D-antigens that are inherited
as a block is missing in any antigens it is known as D-mosaic: also weaken expression of D-
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
antigens may sometime occur in form of D u phenotype. It could be D negative or a weakened D
when typed with high protein potentiators or chemically modified D-antisera. In addition to the
above there are also different subtypes of each D antigen such as D w or D well (as R23) etc. the
other anti-D antibodies and the subtypes of D-antigens are out of the scope of the rest of this
topic and need to be referred to other tangible infos available in other sources. Table 2-7 shows
the compound Rh antigens and their correspondent haplotypes (Reid et al, Daniels G et al and
Quinley et al.)).
Table 2-7: Compound Rh antigens and their correspondent haplotypes
Cmpd antigens
Ce(rh i )
cE
ce (f)
Haplotypes
CDe(R 1 ) & Cde (r´)
cDE (R 2 ) & cdE (r”)
cDe (R 0 ) & cde ®
CE
CDE (R z ) & CdE (r y )
ce s (V)
cDe s (R 0s ) & cde s (r s )
*Haplotypes are responsible to producing of the different Rh antigens.
In this regards Rh o (D) is the most important and significant in Rh system (in a non-ABO group).
It can be positive or negative in the individual. Anti-D is formed by alloimmunization of the
mismatched donor’s Rh phenotype or by feto-maternal alloimmunozation. It is one the
most important causes of HDN, next to ABO incompatibilities. Once the Rh positive cells
combine with anti-D due to IgG properties it may lyse the red cells (Rh+): the condition can
jeopardized the fetus with hyperbilirubinemia (prehepatic jaundice/the HDN is classically
known as” icterus gravis neonatorum or erythroblastosis fetalis”..
Rh anitbodies (anti-D) is an IgG antibody class with usually IgG 1 and IgG 3 subclass, these
antibodies cross the placental barrier due to their low molecular size and if positive for Rh (+)
can hemolyse the red cells in utero causing HDN. The treatment consists of phototherapy with
certain UV wave length and or exchange transfusion in severe cases post-delivery/post-partum.
(with bilirubin more than 300 μmmol or 18 mg/dl). Above this level of bilirubin the child may
experience Kernicterus which is demyelination (myelinolysis) of the neurons. The high
bilirubin level may transgress through the blood-brain barrier and inflect the injuries to the brain
and compromise the child’s survival, or it may survive with a brain damage. The fetus is not
affected by high level of bilirubin in utero because of mother’s liver handling the toxicity of the
unconjugated bilirubin (hyper-bilirubinemia) however post- delivery neonates due to
immature liver as said may experience fatal increase in amount of bilirubin and rapid medical
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
response would be essential. In addition for management of HDN in the subsequent pregnancies
the anti-D immunoglobulin (Rhogam) will be injected IM (intramuscularly) to the mother at
antepartum dose at 28th weeks of gestation and or at 72 hours post-partum dose. Figure 2-3
shows the HDN event in the fetal-maternal hemorrhage (FMH) and Rh incompatibility in
utero and during delivery: when the umbilical cord is severed it may be compromised adding to
fetal maternal hemorrhage and incompatibility between mother and the child.
Fig. 2-3: Indications for HDN/erythroblastosis fetalis/icterus gravis neonatorum as Rh
incompatibility between maternal and fetal circulation with IgG class 1 & 3. These can pass
through placental barrier and affecting the fetus in vivo: for more information refer to the text.
Other Blood Group Systems (Daniels G et al & Quinley et al)
Subsequent to discovery of ABO and Rh system by Karl Landsteiner and others the studies on
blood group system become more intensified and the fruit of these researchs led to discovery of
more and more blood group systems. Currently there are about 600 blood group antigens
today in comparison or 3 in 1900. The following systems are the representative of these groups
of red cells antigens in brief. For details of the systems refer to sole immunohematology and
transfusion medicine textbooks or other sources.
Lewis System
Lewis system antigens are presented by the symbol LE with three phenotypes of Le (a-b+), Le
(a+b-) and Le (a-b-) seen, however the phenotype Le (a+b+) are usually seen in children than
adults and are rare. Neonates usually have Le (a-b-) Lewis blood types after birth with
conventional blood typing and matching methods although more advanced method had shown Le
a
antigen on their red cells after the 2nd months of birth. The different detections patterns at the
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
2nd month has shown that at this age neonates may show blood type Le (a+b-), and Le (a-b+) at
2-3 years of age and Le (a+b+) during sometimes at post-delivery to 2-3 months.
Lewis antigens are not intrinsically produced by the red cell on their surface, however it is
present in plasma and secretions as I antigen, in this way they would adsorb to the red cell
surface. The expression of Lewis antigen system is under control of Le and le gene loci
nevertheless, Hh, Sese, sese (secretor genes) do as well control the production of the antigen
phenotypes.
Lewis (LE) antigens are present under certain circumstance such as: Le (a+b-) is coded by the
presence of at least sese secretor genes, H and Le genes. Accordingly the Le (a-b+) phenotype
encoded as well by the presence of Le, Se and H genes and may manifest as Le a , Le b
antigens in the secretions. There is also Le (a-b-) phenotype that may be coded by the
availability of a lele, se and H genes. In addition, the presence of lele, Se and H genes may
codify/encode Le (a-b-) as well.
As result of the Le, le, Se, se and H gene rearrangement, there are different patterns of red
cell phenotypes.
Other Lewis antigens include Le x , Le c and Le d with other compound antigens as A 1 Le b and
BLe b . Le x antigen is antigenic and agglutinates cord blood cells of neonates who inherit Le
antigen and are typed Le (a-b-). Other antigens such as Le c and Le d may react differently with
Le (a-b-) phenotype on secretor and non-secretor bases. The following table (2-8) shows LE
phenotypes and testing with panel of reagent O.
Table 2-8: Phenotypes and group O cell panel responses to LE
antisera
Reaction with
Anti-Le a
+
0
0
+
Anti-Le b
0
+
0
+
Phenotype
Le(a+b-)
Le(a-b+)
Le(a-b-)
Le(a+b+)
Compound antigens have antigenic activity of blood group A 1 or B and Le b antigens. With
regards to Lewis antibodies, it includes anti-Le a , and anti-Le b or other Lewis antibodies.
Anti-Le a antibody is usually IgM class/type as well as anti-Le b . This antibody class is a room
temperature reactive agglutinin capable of complement binding and activation. Anti-Le a may
be provoke/produced against Le (a-b-) phenotypic red cells. The Le a antibody may as well
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
be partially or totally of IgG class or type. The major reactivity may be seen at room
temperature or sometimes at 37°C (IgM and IgG classes). Anti-Le a and Le b are not capable of
developing HND (Hemolytic Disease of the Newborn), nor HTR (Hemolytic
Transfusion Reaction), however HTR has been documented in some examples of Le a . Both
antigens testing may be done by using monoclonal and polyclonal antisera and reagents (for
Le a and Le b ).
I blood group system
This system of red blood cell antigens is associated with autoantibodies. I antigen agglutinin
are not known to cause a HDN due to anti-I IgM antibody (this antibody class cannot cross
placenta), however anti-I IgG has been documented to have caused on one occasion HDN.
Antigens of I blood group system include, I and i antigens whereas I adult frequently associated to
adult type of individuals. However I int ermediates possesses equal strength of I and i antigens on the
red cells, and also i 1 and i 2 have more and a stronger amount of i antigens on their red cell
surface and there is a weak strength of I on red cells.
Usually anti-I and anti-i antibodies are of type IgM or class and cannot mediate destruction of
red cells as it cannot cross the placenta however enhance destruction as said earlier for IgG
class has been documented in one occasion. The weak distribution and expression pattern of I
antigen on fetal red cells is a factor to protect the antigen from destruction as well. In addition
the anti-I produced against i- antigen is likely to cause HDN due to rare class of IgG which can
cross placenta. In respect to HTR, it cannot be caused by autoantibodies such as autoanti-I but
only in rare occasion as a strong and potent autoanti-I or i which is associated with hemolytic
anemia (Daniels G and Quinley et al).
Autoanti-I are linked to I antigen is capable of causing cold agglutinin disease (or CAD): this
is a potent example of autoanti-I. As the same autoanti-i is associated with i antigen and can
cause infectious mononucleosis (IMN), it has IgM characteristic. In regards to autoanti-I it is
likely a benign autoantibody encountered in majority of people. Autoanti-I can be detected in
4°C cold agglutinin disease as autoantibody this is totally an IgM antibody. It may react at
antiglobulin phase and binds to complement as it can be eluted (separated) from the cells at
37°C incubation from the red cells, however the complement remains with the antiglobulin
reagent on the surface and may cause lyses of cells to prevent this artifacual destruction and
thus hemolysis: technologist should pre-warm the patient’s serum tube or the slide before testing.
Likely the anti-I is as well a cold agglutinin (as in CAD) as anti-i and it is of IgM class, Anti-i is
linked (associated) to IMN and a benign form of antibody in reticulosis, alcoholic cirrhosis
and myeloid leukemia.
P blood group system
Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
547
P blood group system was discovered in 1927 by Landsteiner and associated (Levine). The
antigen is called “P1” by international society of blood transfusion (ISBT). This complex
system of antigens are divided into the following category: the most common phenotypes are P
k
( and the fourth P 1k and P k2 ) the
1 and P 2 (which is the absence of P 1 ) and the third one is P
fifth is p antigen and lastly soluble P system antigens (this is found in plasma and other
secretions, it is rich in hydatid cyst fluid (P 1 ). The following table (2-9) shows phenotypes
and their reaction with correspondent antibodies.
Table 2-9: The P system phenotypes and the reactions with
correspondent antisera
Rxn with
Anti-P 1
Anti-P
+
0
0
+
0
AntiPP 1pk
+
+
0
+
Phenotype
+
+
0*
0
AntiPk
0
0
0
+
0
+
+
P k2
P1
P2
P
P 1k
*= usually negative or weakly positive.
The P system of antigens is found in on all red cells and all are part of this blood group system.
The antigen system is defined according to their biochemical differences and antigen P 1
classifies to P 1 and P antigens and P 2 individuals may have only P antigen on their red cell
surface. It can produce anti-P 1 .
The inhibitory gene In(Lu) can inhibit the expression of P 1 antigen.
The P k antigen (the third antigen of P system) is divided into two other different antigens
expressed on the surface of red cells these are known as “P 1k and P k2 . In this respect the P k
positive cells always lack P antigen. The p [lower case/it is also known as Tj(a-)] phenotypes
ultimately represent an antigen that lacks all phenotypes (P, P 1 and P k ). Antibodies include
the anti-P 1 , anti-P and anti-p [or known as anti-PP 1 P k or Tj(a-)].
Anti-P 1 occurs in serum of P 2 individuals and is of class IgM (a cold reactive agglutinin) the
antibody is capable of binding complement and therefore lysis of the red cells. It may not react
above room temperature and may not be detected and therefore enzyme technique may help
modify the reaction and enhance the in vitro hemolysis. It may cause HTR rarely.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Anti-P is an alloanti-P is a potent IgG hemolysin and appears in PCH anemia (CAD/proxysmal
cold hemoagglutinin disease/hemoglubinuria) it is produced against “P 1k and P k2 ” phenotypes. It
is a biphasic (cold & warm reacting) IgG hemolysin (warm reacting agglutinin) which reacts
in both 4°C and 37°C temperature, it is known as “Donath-Landsteiner” biphasic antibody.
The anti-PP 1 P k (as the mentioned earlier phenotypes) may be either IgM or IgG hemolysin
and the women with increased p phenotype have spontaneous abortion (which may be linked
to anti-PP 1 P k ). There are other antibodies such as anti-luke and anti-p and other compound
antigens that are of minor importance and have not been covered in here. There are antisera
available by manufacturers that can be used in P 1 soluble antigen-antibody neutralization
(serum neutralized). A saline control substituted for soluble antigen must be used with the test.
Kell blood group system
The ISBT symbol for Kell system is KEL: Kell locus codes for all Kell antigens. This system
is well developed during fetal and after birth on the neonates red cells. The main phenotypes
include K, k, Kp a , Kp b , Js a Js b K 0 and Kx (McLeod phenotype, associated with CGD type
II /chronic granulomatous disease and red cell deformities as acanthocytosis, reticulocytosis
and anisocytosis). These are accordingly given name by ISBT as KEL1, KEL2, KEL3, KEL4,
KEL5, KEL6 and KEL7 respectively. There are other subloci antigens with high and low
frequencies that exist, which usually segregate differently according to their availability. They
pose an allele for high frequency antigens such as k/Kp b /Js b and KEL11 and three allele from
low frequency antigens such as K, Kp a or Kp c , Js a and KEL17, Their frequencies varies
with the race. There are also other antigens named para-Kell antigens which are not
associated genetically with KEL system locus. The examples are KEL12, KEL13, KEL14, and
etc.
Antibodies to KEL system include anti-K: a strong immunogen (next to D), it is immune
mediated IgG antibody reactive in antiglobulin phase. This antigen is able to be produced
anti-K once sensitized for years at a detectable level. It is capable of inducing HTR and HDN.
Anti-K is the most significant of KEL antibody systems and others are less common.
MNSs blood group system (Neel JV et al/Quinley et al)
The ISBT have selected the system name as MNS: the antigen to this system is composed of M
(MNS1), N (as MNS2), S (as MNS3), s (MNS4) and U (as MNS5). These antigens are
glycoproteins and are located on the molecular structures of N-acetylneurominc acid are
commonly called “sialoglycoproteins” or SGPs. These trans-membranne proteins expand
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
through the red cell membrane. MN locus has its own SGPs known as “gylcophorin A” or M
N GPS and Ss have “glycophorin B”, or Ss SGP. The amino acid sequence of the gylcophorin
A M is different from glycolphorin A N , the same applies to glycophorin B for S and s antigens
commonly known as glycophorin B S and glycophorin B s . These are trans-membrane proteins
with their own specificities. U antigen is the third allele located on Ss locus (nominally called S
u
and neither S antigen nor s antigen is present on the red cell. There are other varieties of S u
locus such as S u S u which lacks Ss SGP on their red cell surface (S  s  ). This is a homozygous
state and usually is U negative however U var a kind of S  s  U  phenotype exists and is
positive for U antigen. Other MN antigens are exampled by ‘N’, En and Mk. There are many
other examples of MN phenotypes such as Miltenberger subsystems and M g and Can with
Tm and other which will not be included in here for their antigen and antibody significance.
Antibodies to the MN and Ss and U system which are the most important antigens in this
system include anti-M as a IgG (mostly) or IgM class and usually react below body temperature.
20% of random donors are M-antigen negative. It is recommended to perform the
compatibility test at 37°C to avoid technical problem. Although some of anti-M are insignificant
but nevertheless anti-M antibodies often can cause HDN and may rarely cause HTR at 37°C with
often enhanced patient’s red cell destruction and low survival. Anti-N is a weak cold reactive
agglutinin naturally occurring IgM. This anti-N shows antigen dosage (reacts better with double
dose or homozygous state). This antibody reacts with 7% of the general population (random
blood). The Enzymes destroys anti-M and anti-N activity. Anti-N is not clinically significant
however it may react at 37°C, and the compatibility test to overcome technical problem
should be endorsed at the same temperature (37°C).
Anti-S and anti-s are usually immune antibodies (IgG) and may react at AHG phase
(antihuman globulin). For Anti-S, IgM class may be seen. Anti-s is almost always immune
IgG class. Clinical significant may be HTR inducing and HDN cases have been reported. S
antigen negative blood should be given to patient’s having the anti-S. Anti-s is usually immune
(IgG) however cases have been reported causing HTR and HDN. Anti-s may also be direct
agglutinating cold agglutinin (IgM). Anti-U is also HTR and HDN inducing and is of IgG
reacting at AHG phase at 37°C. it is produced by S  s  U  phenotype.
Duffy blood group system
Duffy system presents by short terminology form FY system by ISBT. The major antigen
system in this blood group and the correspondent alleles are Fy a (FY1), Fy b (FY2) and Fy
(Fy0). Fy x (a genetically determined weakly reactive expressed Fy b antigen) , FY3, FY4 up
to FY6 are other antigens and phenotypes of Duffy system. Anti-Fy a is almost often immune
reactive type IgG and is reactive at AHG phase at 37°C: 34% of whites are Fy(a-) and 70% are
black with Fy (a-b-), these are the sources of compatible blood and can solve antibody
problems in compatibility testing. This antibody clinically can induce HTR and a mild or severe
HDN. Anti-Fy b is the same as anti-Fy a as immune antibody (IgG) and is AHG reactive.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Duffy six or FY6 [Fy(a-b-)] acts as the red cell receptor for Malarial parasite, Plasmodium
vivax. The FY1 and FY2 are causes of HTRs and may cause mild to severe HDN. In these
cases blood should be transfused that lack Fy(a-) and Fy(b-) antigens/phenotypes.
Lutheran blood group system (Daniels G et al)
The ISBT terminology used is LU and there are four antithetical pairs of antigens coded by
alleles at closely linked genetic loci. These antigens are Lu a and its antithetical Lu b and the Lu
ab
and respectively are numbered and coded by ISBT as LU1, LU2 and LU3. There are genetic
linkages between Lu and Se (secretor) genes/loci on chromosome 19 (this is an autosomal
linkage). In addition, there is also several associated but unrelated para-Lutheran high- and
low frequency antigens that have been reported. The anti-Lu a is IgG class and is reactive with
AHG and sometimes may agglutinate the red cells directly [Lu(a+)] and may demonstrate mixed
field reaction or agglutination (this is when some agglutination is found among other unagglutinated red cells). It is not a cause of HTR however it may induce a mild HDN. It is
important that the antigen has only 8% frequency in the general population therefore the donor
blood units that have strong reactions with the antibody in compatibility testing should be
issued
and be taken as a precautionary step to avoid likely complications. Anti-Lu b is IgG immune
antibody with reaction in AHG phase the frequency is very low and can reduced red cell survival
and may be a cause of HTR: a mild HDN cases have been documented. Anti-Lu ab (Anti-LU3)
is the same as LU2 and LU1 it is an IgG immune antibody which reacts with AHG at 37°C. It
is present in the serum of people with recessive phenotype Lu(a-b-) and has been implicated
in HTR and HDN, nevertheless the reactions are of delayed type.
Kidd blood group system
The international society for blood transfusion (ISBT/International Society of blood
Trandfusion)) named the system as JK. The most important antigens and phenotypes in this
blood group system are Jk a (JK1), Jk b (JK2) and a null phenotype such as Jk(a-b-). There are no
high- or low frequency antigens and the system is quite straightforward. Anti-Jk a is the
most dangerous immune IgG antibody that occurs in some patients through feto-maternal
immunization (hemorrhage/etc.) of transfusion, it reacts with polyclonal AHG and fades in
vivo and vitro. It binds complement, It may cause a brisk hemolytic reaction in incompatible
blood is given this might be detected due to unavailability of sensitive technical reagents for
JK1 weak antibodies. . The ability of the antibody to fade and evanesce after incompatible
transfusion/immunization is a drawback to technologist performing the compatibility
testing/antibody screen. This antibody disappears quickly after sensitizing transfusion, For
incompatible blood Jk(a-) blood must be issued when patient is screened positive for anti-Jk a .
About 23% of random donors are Jk(a-). This antibody causes immediate or delayed
transfusion reaction and is a ground for sever hemolytic reaction and red cell
destruction/hemolysis. It may induce HDN as well. Anti-Jk b is also an IgG immune antibody
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
reactive at polyclonal AHG stage. Its clinical significance is the same as anti-Jk a : it is a cause of
immediate and delayed transfusion reaction and may cause HDN as some cases being HDN.
For anti-Jk b antibody positive in patient’s serum, the blood unit must exclude Jk(b-)
antigen/phenotype. About 26% random donors are Jk(b-). Jk3 antigen is coded by the two
loci/genes that code for Jk a and Jk b . Anti-Jk3 is an immune IgG antibody and best react with
poly-specific AHG antisera/reagent. It is mono-specific and is not a mixture of anti-JK1
and JK2. Some people may produce this antibody to their red cell phenotype Jk(a-b-) or called
JK3. It is also distinguished by its resistance to lysis of 2M urea.
Other Blood group systems
There are other blood groups systems that are important in transfusion medicine however,
some of them will be shortly covered here. In these regards Diego system is composed of Di a
and Di b antigens with system number is 010 and system code name by ISBT is DI. Anti-Di a
and anti-Di b and their related antigens and phenotypes are used to study geographical
distribution and migration pattern between populations. About 36% of native-Americans
and south-American and Asians is Di (a+). Clinical significance of these antibodies are the
causes of HTR and HDN. In addition, anti-Di b similar to anti-Di a could be involved in red cell
hemolysis and destruction of these cells as in HTR and may cause HDN.
Next blood group system would be Xg located on the X-chromosome, additionally, there is
only one antigen as Xg a (XG1) in this blood group system and two phenotypes Xg(a+) and
Xg(a-). ISBT system name is XG and it binds complement as well, with no in vitro hemolysis.
It is not clinically associated with either HTR or HDN.
The last blood group system would be Dombrock system: known as DO by ISBT and with the
numeric code and number designation of 014. It consists of two antigens Do a and Do b . It has
three high-incidence antigens Gy a (Gregory), Hy (Holly) and Jo a (Joseph), these antigens are
distributed evenly in general population. Both antibodies are IgG immune as well and anti-Do
a
can cause HTR and in some cases have proven to be able to induce HDN; anti-Do b is a cause
of HTR nonetheless it has not been documented in HDN. Other blood group system are out
of the scope of this text and is not meant to cover all aspects of transfusion medicine, so for
acquisition of tangible amount of information in regards to transfusion and blood group
systems consult the references at the end of the part four.
Chapter three (3)
Direct Agglutination Test (DAT/Coombs’ Test)/(Quinley et al/Klein et al)
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
T
his test is used to detect in vivo sensitizing antibody (-ies/proteins) coating the red
cells. It is performed by spinning and washing (or no washing) the blood obtained from
the patient suspected of having antibody to the red cells. If the suspected IgG or C3d
proteins can be demonstrated on the patient’s erythrocytes, it will indicates presence of
autoimmune hemolytic anemia (AIHA), hemolytic anemia, HDN (Hemolytic Disease of
the Newborn) and Drug Induced Hemolytic Anemia (DIHA). After spinning the blood and
washing (or no washing), the spun washed blood with %2-5 suspension (of red cells), may be
tested with AHG serum (antihuman globulin/Coomb’s reagent), if we observe any
agglutination the test is positive and the strength of the reaction is noted. Subsequently, a further
investigation may be needed: in some cases it is essential to identify the protein(s). For example
in autoimmune hemolytic anemia the above components of IgG with or without C3dg may be
found on the suspected red cell(s), however in not all cases the antibody ID is indispensable by
eluting. The presence of antibody on the red cells after the investigation will not point to the
exact etiology or the cause of disease (e.g. HDN, AIHA or DIHA), nevertheless indicates an
underlying process. The main difference between the DAT and IAT (indirect antiglobulin
test) is that in the procedural protocol there is a component of incubation at 37°C after the of
AHG addition in the IAT whereas it is not included in DAT. There may be some false positive
or false negative results. False positive may be indicated by the technical errors, personnel
erring, sample contamination, or inappropriate blood sample. False negative may be seen in
failure to add antiglobulin reagent/serum, improper technique, under-centrifugation and etc.
The following figure (3-1) shows DAT principal.
Fig. 3-1: schematic presentation of DAT in which incubation at 37°C has been omitted.
Indirect antiglobulin test (IAT/Indirect Coombs’ test)
The IAT is performed to detect unexpected antibody and its identification, compatibility
testing, antibody screen, red cell elute testing and antibody titration The procedure is the
same as DAT with an incubation component at 37°C after the AHG serum addition. It detects
antibody in the patient’s serum as it is sometimes called antibody screen. It is a part of type
and screen procedure. %5 of people is usually positive for IgG or IgM antibodies and or both. If
the patient is transfused or being pregnant and sensitized with antibodies mostly would be of IgG
class (immune reactive). These antibodies (IgG) will react best with reagent at 37°C and AHG
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
will help to make a bridge between the incomplete antibodies allowing them to agglutinate
antibodies on the red cell more ambiguously. Incubation at 37°C helps to increase chances of
agglutination between the AHG serum and the incomplete antibody on the red cell surface.
(e.g. antibodies to Kell, Kidd, Duffy and Rh systems). IgM antibodies usually are not significant
except in cases of ABO incompatibility, However IgM class (e.g. I, P, MN antigen systems)
may cause some difficulties in serology in vitro practice and should be takled efficiently. IgM
is saline reactive (using LISS/low ionic salt saline solution) and reaction occurs below
room temperature down to fridge temperature. In IAT testing, serum of the patient is incubated at
37°C with AHG along with the red cell reagent for a length of time as 20 minutes and the
reaction will be noted either as positive or negative. Positive result often mandates a further step
for identification and specificity determination of the antibody detected. The most advance
testing for DAT and IAT uses gel test, SPRCA IAT (known as solid phase red cell adherence)
and ELISA (enzyme kinked immune-sorbent assay).
Pre-transfusion testing (Klein and Quinley et al)
Pre-transfusion testing regularly consists of ABO forward testing or typing; next will be Rh
typing, third ABO reverse grouping, antibody screen (which is usually IAT) and lastly crossmatch. Shortly the ABO forward grouping can be performed by recipient red blood cell with
anti-A and anti-B. Rh typing necessitates the use of patient’s/recipient red cells with Rh
antiserum along with an Rh control. In addition the ABO reverse grouping may be done with
recipient serum (plasma) with A 1 cells and B-cells. With regard to antibody screen recipient
serum (plasma) may react with screening cells. And lastly the cross-match consists of recipient
serum with donor red blood cells. Compatibility testing is referred to cross-match which is
the only one element used in pre-transfusion testing in current use.
Before the testing are done the documentations such as request forms, requisition, patient’s
identity and collection of the blood sample are part of the pre-transfusion workups (a current
sample is essential to resting). In testing the strength of the reactions such as 4+ to 1+ and +/- to
0 must be documented and reported. Among test done on patient in pre-transfusion services
and in any other services is ABO compatibility. In regards to type and screen, this is when the
physician requests the “type and screen”. This include ABO/Rh typing and antibody screen
(IAT/Indirect Antiglobulin Test). During any testing the visible sign of antigen-antibody
reaction are hemolysis and red cell destruction of low survival cells and these should be
considered “a positive” for agglutination or hemolysis and therefore incompatibility between
the recipient and the donor’s unit. In antibody screen the screening cells antigenic profile include
D, C, c, E, e, Kell, k, Le a , Le b , Jk a , Jk b , Fy a and Fy b , P1, M, N, S and s, which are the
genetic profile of the individual donors as screening cells. These are used in antibody
screening and compatibility between the recipient (vide infra) and the donor cells. The last stage
is the cross-match which is done between recipient serum and donor’s blood cells it can be
minor cross-match or major. The minor cross match has less use and not recommended for
current used due to its lack of usefulness. The major cross-match includes patient’s serum and
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
donor’s blood unit. The technique usually is the same as IAT or antibody screen. We may
include immediate spin and IAT in most cases of cross-match.
Type and screen
In type and screen the ABO/Rh blood group and antibody screen (IAT) may be done on request
by physician for “type & screen”. This consists of screening by screening cells (vide supra) as
mentioned already and a forward and reverse ABO grouping. The Rh antigen is tested by
antisera for Rh (D). Weak anti-Rh may be determine with antisera to D u antigen.
Immediate spin cross-match can be used on the patient’s serum (recipient) and donor’s blood
unit. It is good to determine the IgM of the ABO antigen/antibody reaction, once the serum and
red cell suspension (2-3%) has been done, dislodge the cell button and observe either under
microscope or visually the agglutination/clumping or hemolysis of the mixture. The antibody
screen usually utilizes the application of antigrams (e.g. 3-2) and cell screens, the example of
which is indicated in the following figure. For complete application of antigram investigation
and processing consult immunohematology and transfusion medicine texts as in Eva. D. Quinley
and others.
(A)
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
(B)
Fig. 3-2: (A-B) examples of antibody screen with cell panel/screen in two different computerized
and non-computerized antigrams.
Types of donation and component transfusions (Eichiert I et al and Sutton RG et
al)
These are divided to homologous donation, autologous transfusion, preoperative salvage,
intra-operative salvage and post-operative salvage.
In homologous transfusion the patient’s given homologous blood donated by other
individuals of the same species (human).
In autologous transfusion the patient’s own blood will be transfused during operation or
surgery: in this type of donation the patient’s blood unit have been taken and collected prior to
surgery.
In preoperative autologous donation the patient’s own blood is collected prior to surgery as
mentioned earlier: patients with risk associated to it such as patients with valvular stenosis and
unstable angina due to coronary artery insufficiency are not accepted for donation. Patients
with cerebrovascular accident or insufficiency may not be a candidate for autologous donation
for these autologous donations may induce hypovolemia in the patient. The recipient who
donate his/her own blood must not have bacteremia as well so that to disseminate it and produce
autoinfection and worsen the condition. These patients are proven to more morbidity and other
patients.
In intraoperative salvage (Freischlag et al) the patient is given his/her or an autologous
donation during surgery: the collected blood during operation may be collected by devices and
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washed and returned/infused back to the patient as needed. In intraoperative salvage the blood
lost in and during surgery is collected, and infused back to the patient. This is the major autotransfusion procedure considered due to the risk associated with allogeneic (separate individual)
transfusion. Therefore given a lot of attention during the past and present usually it is used in
cardiothoracic and vascular surgeries. In this salvage devices to wash cells (cell washers
and saviors) and process the red cells are employed.
In post-operative salvage the blood lost through post operation bleeding is collected washed
by cell washers and saved and subsequently infused into the patient back as needed.
Hemarthrosis and closed operative wounds may be managed and the lost blood collected
and re-infused into the patient for the purpose of increasing the volume and hemoglobin and
tissue oxygenation status of the patient. Patients with major blood loss and inadequate
oxygenation during surgery such as cardiothoracic (cardiopulmonary insufficiency and
coronary by-pass grafting surgery) and orthopedic surgery patients who lose blood during
post-surgery event (up to 400-500 ml) may benefit from post-operative blood salvage. The
salvage system and devices operate on the bases of direct infusion, washing, processing and
saving along with ultrafiltration of blood constituents. The disadvantage of the salvage is that if
the wound site or operative site be infected or cancerous there may cause dissemination of
infection and metastasis however there is no actual documentation of such disadvantages.
Side effects and adversities of transfusion (Quinley et al)
There are numerous adverse/side effects with transfusion therapy this may include but not
limited to the following. The effects include Intravascular transfusion reaction is one eminent,
which can occur by incompatible blood unit transfusion . The antigen (blood group
systems) and antibodies especially IgM type for immediate transfusion reaction can ensue in any
transfusion event, if the compatibility and safety has been compromised by erring in screening
and compatibility testing. This is when the complement and kinin system have been activated
which ultimately results release into circulation in form of C3a and C5a acts as anaphylatoxins
and can be released in the intravascular compartments by accumulation of leukocytes
(polymorphonuclear leukocytes) and vasoactive amines along with histamine release. This
will cause coagulation derange causing DIC (disseminated intravascular coagulation) and related
thrombocytopathies (thromboplastic substances) which may be a drastic event to follow.
ABO/Rh grouping and other related transfusion strategies must proceeds by SOP guidelines
(standard operation procedures) to prevent transfusion adversities. Next is a delayed
extravascular transfusion reaction, which is more likely with blood groups JK and Duffy, Kidd,
Rh, P and MNSs are the culprit in this scenario. The C3b activation is activated only up to this
stage and type of antibody involved is IgG and immune mediated reaction: delays usually last up
to 7-14 days due to anamnestic response.
Other factors attribute to the adversities are potassium imbalance/hyperkalemia (shifting inside
the cells) due to hemoglobin and circulatory overload and current RBCs lysis (hemolysis), this
may result to kidney failure due to massive accumulation of red blood cell stroma and
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potassium shifting from lysed/ruptured incompatible transfused RBCs; citrate toxicity;
hypothermia; transfused hemosiderosis; bacterial infection (as with them endotoxins, pyrogens,
e.g. cold growing bacteria, as Pseudomonas spp., Citrobacter ferundii, and Escherichia coli. and
Yersinia enterocolitica) along with osmotic rupture of RBCs. The Post transfusion purpura may
be observed in patient who have platelet antigen PI A1 negative and most experience an anti-PI A1
with anamnestic response due to IgG class antibody to the platelets. Other adversities of blood
transfusion are infection, sepsis and bacteremia and other diseases transmission, such as
Salmonellosis, Trypanosomiasis (T. cruzi); malaria and filariasis/microfilariasis, HIV and
hepatitis B, and C. Diseases transmission by transfusion will be covered partly in diseases
transmitted by transfusion in the section next.
Diseases transmitted by transfusion (Slichter SJ et al)
Here there are numerous numbers of diseases that can be transmitted by horizontal transfer
pattern. These patterns include also transfusion transmitted diseases as described here. This
covers, viral infections, HIV, Hepatitis viruses (A, B, C, D etc./ chronic hepatitis.), syphilis,
CMV, IMN (Epstein bar virus/EBV), Parovirus, Prions ( as in Creutzfeldt-Jakob
syndrome). Also bacterial infections such as Lyme disease, Yesinia enterocolitica, undergoing
bacteriemia/sepsis, along with parasitic infections as Leishmaniasis, Chagas disease
(Tryponomsoiasis), toxoplasmosis, babesiosis and malaria may be the important causes of
transfusion transmitted disease, however most of these infections and diseases can be screened
prior to infusion of the blood unit to the recipient with some exception which as a matter of fact
need a thorough clinical history of the donor and other detection applications.
Out of these myriad of diseases transmittable by transfusion most important are HIV and
hepatitis viruses. I briefly explain the cause and the pathophysiological stand point of each
disease in short including serological test if and when available.
HIV virus: a retroviridae family as HIV-I an HIV-II, these affect human lymphocyte T4
(helper-T lymphocytes) by mimicking these cells genomic code/profile by a polymerase like
enzyme called reverse transcriptase. Thus in this way the replication and proliferation can
reach to critical numbers then burst the host cell and kill them (T-cells), once the host genetic
code changed the virus buds and destroys the T4 cells , these seems to be the tone of
mechanism and mode of operandi of these virus and its pathophysiologic mechanism. This is
highly infectious once reach to the blood stream and during this period it can be transmitted by
the blood unit and other blood products. Clinical history and confidentiality of information of
are of prime importance in donors with the HIV illness (see CUE/or confidential unit exclusion):
this is a base to deferral. Once T4 cells deplete the patient immune apparatus emaciates and
patient succumbs to it by super- and auto-infections. Serology tests consist of EIAs (enzyme
immunoassays). Antibody to the somatic virus persists after 3 month and can be detected
therefore EIAs may be of great used in this a viral lysate would be obtained (recombinant or
synthetic antigens) and coated on the microtiter-plates, the test proceeds to an enzyme
labeled conjugate antihuman serum antibody while added and incubated. The other tests
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
such as Western blot and PCR (polymerase chain reaction) is also achieved in identifying
the viral antigens and the genetic profile, the screening of protein; P24 antigen is crucial in
detection of this virus.
With regatrds to hepatitis viruses include C and B and D these are the most significant
hepatitis viruses engage in transmission, nevertheless the hepatitis A and non-A and B viruses
also play critical role in infection and transmission through blood units and blood by-products.
Hepatitis B virus is a double stranded DNA particle known as “Australian antigen” it consists
of out coat of the virus HBsAg (serum hepatitis) called “Dane particle”, this is the first antigen to
occur in the serum during infection. The virus is infectious when the patient’s serum contains
HBsAg and HBeAg. Stands alone anti-HBcore antibody in the serum shows the window period
during which no core antigen is detectable and this is the first viral antibody to occur in
propositus/patient’s serum. The last antibodies are anti-HBeAg, anti-HBsAg to occur as in the
remission stage. The virus is shown in drugs users, and homosexuals, transmission is by blood
and blood products. The detection of this virus by means of serology test such as ELISA and
EIAs are method of choice in screening of hepatitis antigens and antibodies. There are vaccines
(HB vaccine) available to prevent future infections and immunization. Hepatitis B virus
ultimately affects hepatocytes causing eventually cirrhosis of the liver and hepatic cancer
(hepatolenticular carcinoma of the liver) along with fibrosis and necrosis.
Hepatitis C virus the most crucial virus which we have little control on its transmission due to
available serologic methods of detection on routine bases, therefore it is relied and based on
clinical history of the donors and other inadequate tests. It is a single stranded RNA virus with a
capsid (outer envelope) it spreads and disseminates by means of blood and blood products (akin
to HBV) and parenteral exposure and sexual contacts/intravenous drug users. It causes jaundice
and liver cirrhosis, alike to HBV the liver enzymes such as ALT (alanine amino
transferase) may augment and chronicity of the liver cell eventually transform into cirrhosis in
which approximately 10% of the affected patients may acquire cirrhosis (this is an end stage
liver disease) and there may sustain cancer of the liver. Serologic test is based partially on
detection of single protein such as c100-3 and three other generation test antigens as c22-3, c1103 and c33c. The performance of these tests may reduce the incidence of HCV transmission
through blood donation. The three generation tests are more sensitive and reliable. Elevated ALT
alongside this maker with anti-BC detection is to monitor the liver damage progression and has
clinical correlation with the HBC infection.
In addition to the above hepatitis: HAV (hepatitis A virus) is an RNA virus and usually infects
through feco-oral route and rarely transmitted by transfusion, if only the Patient is in the
incubation period. The infection mostly spread by poor hygiene and sanitation and in
populated areas such as nurseries, military camps and hospitals and detention centers. Pervious
exposure immunized the patients and people are not sensitive to the virus by blood
transmission route for most they are immunized as earlier pointed.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
Most of diseases mentioned above may be transmitted through transfusion these are cited in
microbiology part three of this text book. These infectious agents are detectable and screened
during prior pre-transfusion testing and processing of the blood unit and products. (Apheresis
units): example of these include CMV (cytomegalovirus) infection, screening tests have
composed of complement fixation, indirect hemagglutination and ELISA, RIA
(radioimmunoassay) and some others sensitive tests. Epstein bar virus, Prions (as in CJD/
Creutzfeldt Jakob Syndrome/Disease) and other diseases such as above parvovirus infection
including the rest need careful medical history, and laboratory testing may help to maintain
a safe blood unit for infusion and for the safety of the recipient.
Chapter Four (4)
HDN/Autoimmune Hemolytic Anemia and Drug Induced Hemolytic
Anemia (Quinley et al, Slichter et al and Rodgers GP et al)
H
DN (hemolytic disease of the newborn) was previously mentioned briefly: it is also
known as “erythroblastosis fetalis or icterus gravis neonatorum, which is a cause of
hydrops fetalis (neonatal edema). Antigen-antibody incompatibility between
mother and the child is the cause of such sometimes fatal phenomenon. When fetus inherits an
antigen such as ABO/Rh type from father and the mother possess a different antigen from
child/or the father incompatibility exists and mother forms antibody against child’s inherited
antigen: i.e, when father and the child are B+ and mother is O-. The mother forms an anti-Rh
toward the fetus. This eventually crosses the placental cord due to being an IgG class antibody
and lyse the fetal red blood cells of type Rh +. In uterus the child develops jaundice however
remain unaffected due to the most bilirubin are handled by mother’s liver, post-delivery due to
feto-maternal hemorrhage (FMH), child priming and sensitization is much severe and may
experience severe hyper-bilirubinemia and jaundice and may be a stillborn or if there is no
exchange transfusion may experience Kernicterus, this may occur if the bilirubin level exceeds
18mg/dl. The child’s liver is unable to conjugate the bilirubin (clinical chemistry part one/liver
function tests as in pre-hepatic jaundice) produced due to lyses and incompatible red blood cells
and may demise. The antibody lyse the fetal cells and its bone marrow try to compensate with
more immature RBCs hence erythroblastosis (fetalis) term may signify the condition. The
mother must be immunized at 28 weeks of gestation antepartum with 300 μg IM (intramuscular)
injection and or if delivery has taken place the immunization must be done 72 hours postpartum.
In this regard ABO incompatibility may induce HDN as well whereas the mother my possess
IgM class antibody for incompatible ABO blood groups: this is such as child being grouped A or
B and the mother is group O (partially this O antibodies are IgG class), therefore the possibility
of ABO incompatibility exists. This is the most common form of HDN. With Rh system the antiD is the most common form of incompatibility among Rh antigens.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
In respect to testing and screening: automated system for blood group analysis can assay and use
ADCC (Antibody Dependent Cytotoxicity), Monocyte Mononuclear Assays (MMA): these
functional assay systems (non-invasive) estimate the interaction of sensitized RBCs and human
mononuclear cells, in this way the anti-D measurement can be estimated. Ultrasonography and
PUBS (Percutaneous Umbilical Cord Blood Sampling) has improved and is reliable for
monitoring alloimmunization in mothers during pregnancy. Therefore the functional assays
with regards to the former tests are questioned. Other test to monitor the course of pregnancy in
which there are severe incompatibility between mother and fetus is the use of ultrasound,
amniocentesis materials (amniotic fluid) and fetal blood sampling. In this regards, noninvasive method are recommended, nevertheless the amniocentesis (invasive procedure) and
ultrasound are accurate and precise although carry risk to the fetus such as infection, trauma, and
intrauterine demise.
All these tests mentioned earlier help to assess the severity of HDN.
To quantify feto-maternal hemorrhage (FMH) is by use of tests such as rosette test which is a
qualitative test to see agglutinations and mixed-field reactions and followed by Kleihauer
Betke (KB) that is a semi-quantitative test to number the vials of RhIG (anti-D antiglobulin)
needed for use in HDN incompatibility.
Treatment consists of intra-peritoneal intrauterine transfusion (by ultrasound guidance) or
phototherapy (photo-degradation/photo-oxidation and photo-isomerization) in case of severe
postpartum hyperbilirubinemia (associated with neurotoxicity) and anemia, and as well as
mentioned, if the bilirubin exceeds 18 mg/dl there may be a need of exchange transfusion, and
medical intervention, despite of any response to phototherapy neurotoxicity may manifest in
form of passage of bilirubin through blood-brain barrier and affects the neural ganglia
(neurotoxicity) causing Kernicterus. In case of survival at this stage child may experience
brain damage and mental retardation. The ABO/Rh blood typing and IAT must be done for the
fetus or the neonates to qualify as the candidate for intrauterine and exchange transfusion if
blood needed.
Autoimmune Hemolytic Anemia (AIHA)
IHAs (Immune Hemolytic Anemia) divide into two subclasses, 1) Warm Autoimmune
Hemolytic Anemia (WAIHA) and 2) is the Cold Agglutinin Disease/syndrome (CAD) or
cold autoimmune hemolytic anemia. In this type of anemia an autoimmune antibody is provoked
to an intrinsically altered antigens in these cases red cell surface blood groups or antigens. The
antibody would combine with the correspondent antigen (altered self-antigen) and with the
action of complement either lyses the cell resulting into anemia or covers the red cell surface to
be taken into the reticulo-endothelial system for further handling such as destruction or
removal by the said system still ensuing to anemia (Rodgers GP et al/Quinley et al).
The warm type has antigenic correlation and specificity with some red cell antigens such as e, c
or more and frequently (70%) with Rh locus and determinants with no specificity. Practically
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these autoantibodies do not cross react with Rh null . Other specificities as a culprit in WAIHA
induction are anti-U, anti-LW anti-K and anti-Jk a , etc. Antibody in WAIHA is of IgG class and
is responsible for hemolytic reaction at 37°C and occurs in reticuloendothelial system as in
spleen or liver etc. The major organ handling the IgG primed/sensitized cells (coated) will
eventually reach the spleen and will be sequestered there. The macrophages processes the Fc
receptors on the red cells coated with IgG Fc segment, this finally binds to M theta
(macrophages) and the portion/segment of the red cells removed and then the cells reenter the
circulation with spherocytic character (spherocytes), this is due to macrophages processing of
IgG Fc site on the affected/coated red cells surface. This can lead to spherocytic anemia and
extend of that depends on the amount of red cells affected and the compensatory mechanism of
kidney and bone marrow to replace erythrocytes: this is evidenced by
erythrocytosis/reticulocytosis. The condition (WAIHA) can be primary or secondary to
diseases such as LE (Systemic Lupus Erythromatosus/SLE), carcinoma or lymphoma.
Laboratory investigation consists of DAT, elution and adsorption techniques (vide supra),
and antibody detection and adsorption use autologous and homologous red blood cells and crossmatch. In these adsorption methodologies the patient’s own cells are used for autoabsorption whereas in homologous type, group O red blood cells may be utilized when there
are not enough red cells for auto-adsorption or if the patient’s has recently been transfused.
Techniques used in auto-absorption and elution have been mentioned already however
additionally the ZZAP reagent and heating at 37°C can be employed as well to remove IgGs
from the red cells and detect the antibody specificities. Treatment can be used conventionally
by employing glucocorticoid steroids and splenectomy to suppress the immune reactivity.
Clod Agglutinin Disease or (CAD) (Rodgers et al and Quinley et al)
CAD is a hemolytic disease caused by autoantibodies that are autoimmune IgM in nature and
with specificities to I, i antigens. These and other blood group autoantibodies to antigens such as
D antigen, Pr and H are associated with primary idiopathic CAD and secondary to
lymphoproliferative disorders and infections. The antibody is a cold agglutinin means it reacts
below room temperature down to fridge temperature (4°C). The phenomenon of Raynaud’s
syndrome (acrocyanosis) is an example of cold agglutinin formation with subsequent
intermittent attacks of extremities cyanosis and digitals. The red cell lyses in low temperature
due to complement fixation on the red cell surface with correspondent antigen (I,i) and
antibodies (anti-I and anti-i) causing intravascular hemolysis. The hemolysis does not occur
in 37°C or above this temperature (due to saline agglutinin nature of antibodies). In this the
complement combines to IgM (monoconal) and fixes it up to C3 convertase stage and specific
autoantibody may react to lyse the cells in intravascular (intravascular agglutination and
hemolysis) compartment, as the temperature warms up (increases) the C3 components traps on
the red cells to liver combining with their correspondent receptors (CR1 and CR2): C3
convertase splits the C3 toC3a and C3b where C3b on the red cells circulates back to the blood
stream while C3b splits to C3c and C3d: this (C3d) may remain on the red cells and would not be
handle further by the macrophages (M-θ) with normal survival rate. With disease correlation
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the anti-I, demonstrates specificities to Mycoplasma pneumonia infection, the autoanti-I is
associated with CAD (cold agglutinin disease/or CHA/cold hemoagglutinin disease) and
these IgM autoantibodies is polyclonal in nature due to chronicity of the CAD. Relatedly, anti-i
is the cause of IMN (infectious mononucleosis) and other associated secondary illnesses.
Serologic testing protocol consists of DAT and elution. The treatment of choice is the
corticosteroids and or splenectomy on discretion. The autoantibody tested at 4°C either can be
normal or benign with 1:64 titers or at the same temperature may be morbid/pathologic with titer
level reaching to 1:1000 which is significant. The thermal amplitude correlates well with the
intensity of autoantibody and associated better with CHD/CAD.
Proxysmal Cold Hemoglobinuria (PCH)
This is a cold agglutinin disease with AIHA. Patient experience sudden onset of hemolysis
secondary to infection children are more prone to the condition and the disease is associated in
the recent time to acute illnesses occurring in children, it is mostly seen in viral infections such
as M. pneumonia, CMV (cytomegalovirus) infection, H. influenza, and E. coli etc.. The
autoantibody is of class IgG: in this condition both extravascular and intravascular hemolysis
due to release of IgG molecule from red cells and subsequent complement fixation occur
causing red cell lysis, are seen. The autoantibody is known as Donath Landsteiner, this is an
bi-thermic antibody agglutinating at the 37°C and room temperature/or below. Clinical signs and
symptoms may vary from chills, with sometimes high fever, and abdominal cramps, during
the acute attack. Hemoglobin may drop, with hemoglobinouria, with low serum complement
during acute stage and there are hyperbilirubinemia with increased unconjugated bilirubin due
to red cell lysis.
Serologic test consists of DAT and cross match. For detecting Donath-Landsteiner antibody the
Donath-Landsteiner test must be performed in this test the patient’s blood tested in both high and
low temperature (37°C-4°C). A bithermic (biphasic) results in agglutination at both
temperatures indicates PCH. Treatment may indicate a need for corticosteroids therapy and
transfusion of blood must be warmed prior to infusion and splenectomy may not warrant for
might not be helpful in PCH. The disease is a self- limit and alleviates on its own.
Immunohematologic tests include DAT which is positive for complement and the specific IgG
autoantibody is not detectable on the red cells at warmer temperatures and thus elutes is clear at
these (warm) temperatures and is not detected by DAT. Cross match shows specificity for anti-P.
Drug induced hemolytic anemia (Quinley et al)
There are different (vide infra) types of drug induced hemolytic anemia, for a complete account
of this refer to hematology apart three. However the antibody testing for Drug adsorption
mechanism shows the antibody is of IgG class but anti-penicillin IgM antibody variety have
already been documented as well. The DAT is positive with likely IgG antibody or
complement components. IAT is negative with reagent RBCs and usually drug coated RBCs
may be positive with IAT.
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Clinical Laboratory Diagnosis for Clinical Laboratory Scientist
In immune complex or drug dependent antibody type of drug induced hemolytic anemia, the
DAT may be positive and mono-specific AHG indicates patient’s RBCs are coated with
complement components alone. Washing of RBCs may alleviate this by removing any other nonspecific antibodies leaving the complement on the surface: elute should be unproductive.
In the modified membrane mechanism induced hemolytic anemia, DAT is positive and antibody
is of IgG type, red cell membrane is modified and absorbs the IgG and complement from the
serum to RBCs surface, these can be detected on the red cell surface. While in Drug Induced
Autoimmune Hemolytic Anemia, DAT is usually positive, with reactivity with positive
RBC reagents and thus a productive elute. The antibody screen with IAT is positive with normal
reagent RBCs.
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