LABORATORY INVESTIGATION OF TRANSFUSION REACTION CASES
LABORATORY INVESTIGATION OF
TRANSFUSION REACTION CASES
Lecturer name: madam evana kamarudin
Date of submission: 25th october 2013
At the end of this lesson, student will be
- Define transfusion reaction
transfusion reaction occur
- List the preliminary test of transfusion
reaction investigation and its reasons
additional test for the
- Reaction of the body to a transfusion of
blood that is not compatible with its own
- An adverse reaction can range from fever
and hives, to renal failure, shock and
ROLE OF CURRENT DAY LABORATORY
all transfused packs to Blood Bank full
Return giving set and attached solutions
Return post transfusion blood samples
Return transfusion sheet with full details of
WHAT HAPPEN WHEN TRANSFUSION REACTION
Retrieved from : http://www.youtube.com/watch?v=qwu8aqzhgq0
INITIAL MEASURE BEFORE THE INVESTIGATION
Stop the transfusion
An intravenous line with normal saline should be maintained
The patient should then be assessed and supported as necessary while the
patient’s physician and the transfusion service are notified
A responsible physician will need to evaluate the patient and determine
appropriate clinical care
The unit and all tubing should be returned to the blood bank, along with postinfusion blood and urine samples as clinically indicated
The reaction should be documented in the patient’s chart
MINIMUM SAMPLE REQUIREMENT
Verify the patient’s identity using at
least two unique identifiers.
Date and time of sample collection
Ensure all sections in the form are
completed in a legible and detailed
Complete all information in the
“Specimen Collection” section.
Ensure both the Nursing and Facility
of Blood bank clerical checks have
been completed and this is
documented on the form. This will
prevent delays in testing.
Specimen receives unlabeled/improperly
labelled or overlabelled with more than one
Key identifier information is missing, incorrect
or discrepant on the sample and/ or requisition
Specimens not received in the laboratory
within 8 hours of collection
Unacceptable tube received
Specimens which are hemolyzed. Hemolyzed
specimen may contain enough soluble material
to interfere with typing reaction, detection of
clinically significant antibodies or compatibility
testing by acting to neutralize antibodies.
Insufficient of sample. Generally, a minimum of
6 mL of whole blood yielding at least 3 mL of
serum required to provide adequate specimen
volume for antibody and compatibility testing.
The role of lab for hemolytic
Check for clerical errors
Check for visual hemolysis
Test the post-transfusion
sample: Redo ABO grouping
and perform direct
antiglobulin test (DAT)
compare the positive
to pre-transfusion DAT
Post antibody screen
antibody, elution of
DAT (+) cells
Re-do pre antibody
TRANSFUSION REACTION LABORATORY
After the initial measure , the 3 basic preliminary
Purpose : to determine the likelihood the
occurrence of hemolytic transfusion reaction.
If there is evidence of hemolysis or if the clinical
something severe and
unusual, the additional test such as TRALI and
TACO must be performed.
To identify any possibilities of ABO incompatibility.
Compare the component bag, label, paperwork with
patient sample and look for errors.
If an error is found, the physician must be notified.
Most common errors:
Misidentification of patient when pre-transfusion
Mix up of samples in the lab.
Not enough incubation time.
Plasma or serum post-reaction & compare with pretransfusion
This step is done to examine the presence of hemolysis
The destruction of red cells and releasing the free
hemoglobin will resulting a pink to red supernatant
The pink or red colored serum indicate intravascular
Thus the ABO testing must be repeated on the
An urine examination of a post-reaction helps in diagnosis
of an acute hemolysis.
The free hemoglobin in the urine indicates the intravascular
Some causes of false-positive visible plasma
Poor phlebotomy technique (traumatic stick, drawing
through IV line)
Non-immune hemolysis (infusion with 0.45 NS, faulty
G6PD deficiency and hemoglobinopathies
Some causes of false-negative visible plasma
kidneys, hemoglobin may be cleared in several hours)
Sample collected from IV line (dilution of blood)
On post-transfusion sample redo the ABO
test and perform the direct antiglobulin test
sample post-transfusion must be
preserved in a EDTA preservative (lavender
If the DAT is positive on the posttransfusion sample, then one should be
performed on the pre-transfusion sample.
If the result for pre-transfusion DAT is
negative but the result for post-transfusion is
positive, it indicates the presence of
IF ANY OF THESE THREE TEST ABOVE HAVE POSITIVE AND
SUSPICIOUS RESULTS, REDO TEST DONE BEFORE BLOOD
TRANSFUSION WHICH ARE:
1. ABO & RHESUS GROUPING (PRE & POST SAMPLE).
2. ANTIBODY SCREENING (PRE & POST SAMPLE).
3. REPEAT CROSSMATCH (PRE & POST SAMPLE).
REDO ABO & RHESUS GROUPING
ABO and Rhesus grouping are the most important serological test
performed on pre-transfusion.
A full ABO and Rhesus group comprises a forward and reverse
group which must be done at the same time crucial for result’s
Below are table of differences between forward and reverse
Antigen A, Antigen B.
Known Anti-A, Anti-B, AntiAB.
Known A Cells, B Cells, O
More Accurate (Method Of
Accurate, But Need To
Confirm With Forward
No ( ABO Antibodies Is Not
Below are the table summarize the results for forward and reverse
grouping for 4 major ABO blood group.
ABO cells (Reverse)
Anti-A Anti-B Anti- A cells B cells
The principle of ABO grouping is based on a specific agglutination
reaction between antigens on RBCs and antibodies in the typing
+ sign indicate agglutination.
0 sign indicate no agglutination.
2) ANTIBODY SCREENING (IAT)
The aim of antibody screening is to determine presence of the
unexpected antibody other than anti-A and anti-B.
Antibody screening test involve testing patient’s serum against
screening cells which are commercially prepared group O red cells
The cells are selected so that the following antigens are present on at
least one of the cell sample;
D, C, E, c, e, M N, S, s, P, Lea, Leb, K, k, Fya, Fyb, and Jkb.
These are possible reasons why unexpected antibody present in post
Clerical or technical error.
Passive transfer of antibody from a recently transfused component.
Amnestic response : Appearance of alloantibodies can occurs
within hours of exposure (Delayed Hemolytic Transfusion
Antibody screening involve three phases to allow for antibodyantigen agglutination:
1) Immediate spin (Room
• 3 tubes is used using
recipient serum plus saline
suspension screening cell
I, screening cell II, and the
recipient’s own cells for
• Centrifuge these three
tubes and observe for
• This phase detects IgM
antibodies which usually
2) 37°C incubation
• This phase required to
detect the presence of IgG
• Enhancement media is
added such as Low Ionic
Strength Solution (LISS)
• LISS will speeds up
antibodies, so it is add
after immediate spin step.
• Albumin will lower zeta
potential so that cells can
Coombs step and may
detect Rh antibodies.
3) Coombs phase
• This step is important to
detect IgG antibodies,
clinically significant and
capable of causing HFDN
• Wash the cells for 3-4
times after 37C incubation
• Remove saline and add
• Mix and centrifuge
• Read the agglutination
• If the result is negative,
add Coombs Control Cells
LIMITATION OF ANTIBODY SCREENING TEST
This test cannot detect all antibodies of potential clinical
Antibody may be reactive with low incidence antigen
absent on screen cells
If antibody is exhibiting “dosage” it may be missed.
Duffy (Fy), Kidd (Jk) and Rh antibodies may only be
detected with homozygous cells. It will influence
decision to use 2 or 3 cell screen
If antibody screening is positive, additional tests to
specifically identify antibody using the antibody
identification panel and red cell antigen typing must
3) REPEAT COMPATIBILITY
The compatibility testing or cross-match procedure is done again for
confirmation to determine whether blood donor is compatible with
This test involve 3 phases which are Immediate spin, 37°C, and AHG.
The 2 main function of the repeating cross-match test are:
It is the final check of ABO compatibility between donor and patient.
It may detect the presence of an Ab in the patient’s serum that was
not detected in the Ab screening because the corresponding Ag was
lacking from the screening cell.
There are two types of crossmatch :
• The major cross-match involves
testing the patient’s serum with
• To determine whether the patient
has an antibody which may cause
HTR or decreased cell survival of
• This test involves testing the
patient’s cells with donor plasma.
• To determine whether there is an
antibody in the donor’s plasma
directed against an antigen on the
THE CROSS-MATCH HAS MANY LIMITATIONS.
A COMPATIBLE CROSS-MATCH DURING
PRETRANSFUSION WILL NOT:
Guarantee normal survival of transfused RBCs
Prevent immunization of the recipient
Detect all unexpected RBC antibodies in the recipient
Prevent delayed hemolysis due to an amnestic antibody
response to antigens against which the patient has
previous but undetectable immunization
Detect all ABO grouping errors either in donor or recipient
Detect most group D grouping errors in the donor or
TRALI & TACO CASES
Retrieved from : http://www.youtube.com/watch?v=_oQVMcGUwIE
TRANSFUSION RELATED ACUTE LUNG INJURY
Adverse reaction to transfusion that is characterize by hypotension and
Occur when human leucocyte antigen (HLA) or human neutrophil antigen (HNA) antibodies
found in the donor’s plasma are directed against the recipient’s leucocyte antigen.
It is likely to occur to those who were transfused with a large volume of plasma such as
fresh frozen plasma (FFP).
Acute onset of fever, chills, dyspnoea, tachypnoea, tachycardia, hypotension, hypoxaemia
and noncardiogenic bilateral pulmonary oedema leading to respiratory failure during or
within 6 hours of transfusion.
TACO is frequently confused with TRALI as a key feature of both is pulmonary oedema and
it is possible for these complications to occur concurrently.
The main constant feature in TRALI is hypotension.
Chest X-ray showed massive
pulmonary congestion with diffuse
infiltrates for TRALI patient.
A is the normal chest x- ray
image while C is the
imaging of the chest showed
massive pulmonary congestion
with diffuse fluffy infiltrates
Human leukocyte antigens (HLA) and
human neutrophil alloantigen (HNA)
HNA-3a, the former 5b, HLA
class I and HLA class II antibody
indicate severe and fatal cases
For HLA antibody screening,
antibody binding tests (EIA,
preferred- 20% of blood
components contain HLA
HNA antibodies are usually
HNA-3a antibodies which are
known to cause severe TRALI
reactions are often better
detected by agglutination
2. TRANSFUSION ASSOCIATED CIRCULATORY
Adverse reaction to transfusion that is
characterize by hypertension and
This is usually due to rapid or massive
transfusion of blood in patients with
diminished cardiac reserve or chronic
Patients over 60 years of age, infants
and severely anaemic patients are
Dyspnoea, orthopnea, cyanosis,
tachycardia, hypertension and
pulmonary oedema (may develop
within 1 to 2 hours of transfusion)
B-natriuretic peptide (BNP) test
It is a 32-amino-acid
polypeptide secreted from the
cardiac ventricles in response to
ventricular volume expansion
and pressure overload.
BNP levels were measured by
use of fluorescent
In TACO patient, BNP level
Normal value for BNP
0-99 nanograms per liter
3. ACUTE HEMOLYTIC TRANSFUSION
immunologic destruction of transfused
red cells, due to incompatibility of antigen
on transfused cells with antibody in the
Tends to present immediately or within
24 hours after transfusion
common cause is transfusion of ABO/Rh
incompatible blood due to clerical
presence of red cell alloantibodies (nonABO)
in the patient’s plasma which have not
Symptom and clinical finding:
Fever, chills, chest pain or hypotension.
Transfused patients develop
oliguria, haemoglobinuria and
Diagnosis is confirmed by measuring
urinary Hb, bilirubin, and haptoglobin.
Intravascular hemolysis produces free
Hb in the plasma and urine;
haptoglobin levels are very low.
Hyperbilirubinemia may follow.
Male: 13.8 to
0.3 to 1.9
41 - 165
Table 1 :Normal range
4. ALLERGIC TRANSFUSION REACTIONS
Anaphylaxis is a life-threatening allergic reaction that can occur after only a
few milliliters of blood have been transfused
In the case of patients with IgA deficiency, the presence of IgA in the donor's
plasma will trigger for anaphylaxis to occur. Because they lack IgA, their
immune systems develops anti-IgA and sensitize to IgA.
Commonly range from one lesion to widespread urticarial lesions but may be
associated with mild upper respiratory
symptoms, nausea, vomiting, abdominal cramps or diarrohea.
Double immunodiffusion assay
may be used as a screening test to
identify individuals with an IgA level
below 2 to 4 mg/dL.
A more sensitive ELISA method
with a sensitivity of 0.02 mg/dL is
then necessary to determine which
individuals are truly IgA deficient.
Truly deficient individuals, with
levels below 0.05 mg/dL, may
develop anti-IgA antibodies.
Mast cell tryptase test
• The tryptase test is a useful
indicator of mast
cell activation. It may be
ordered to confirm a
diagnosis of anaphylaxis
• With anaphylaxis, tryptase
levels typically peak about
1 to 2 hours after symptoms
• The reference range of
serum tryptase is less than
A small number of bacteria enter the blood
during collection or processing.
During storage, bacteria may proliferate and if
possible produce endotoxin which then will be
transfused to another person.
It is rare but is more often reported with platelet
concentrates (stored at 20-24 C) than with red
cells (stored at 1−6 C).
1. Examination of the pack
Examine: discolouration, smell and gram stain
May rapidly confirm the diagnosis.
2. Blood cultures
Blood cultures from different IV site- to detect any colonies formed on the
streaked agar plate.
3. Product cultures (include a gram stain)
Gram positive bacteria :
Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Group
Gram negative bacteria:
E. coli, Pseudomonas species and other gram-negative organisms.
A type of transfusion reaction that can occur 1 to 4 weeks after the transfusion.
As a result of a secondary immune response with a drop in hemoglobin level.
Usually less severe than acute hemolytic transfusion reaction.
Patients may present with unexplained fever and anaemia usually 2 to 14 days after
transfusion of a red cell component.
The patient may also have jaundice, high bilirubin, high LDH, reticulocytosis,
spherocytosis, positive antibody screen and a positive Direct Antiglobulin Test (DAT).
After transfusion, transplantation or pregnancy, a patient may make an antibody to a
red cell antigen that they lack. If the patient is later exposed to a red cell transfusion
which expresses this antigen a DHTR may occur.
DHTRs may also occur with transfusion transmitted malaria and babesiosis.
Delayed HTR result
Lactate dehydrogenase (LDH)
0.3 to 1.9 mg/dL
41 - 165 mg/dL
Present in urine
Elevated (may be)
Prothrombin test (PT),
Elevated (may be)
Partial thromboplastin time
Elevated (may be)
Free hemoglobin in urine
* D-dimer, prothrombin test (PT), and partial thromboplastin time (PTT) may be elevated, particularly
with disseminated intravascular coagulation (DIC).
TRANSFUSION ASSOCIATED GRAFT VERSUS
HOST DISEASE (GVHD)
A serious complication due to the engulfment and proliferation of
donor T-lymphocytes against patient.
Usually caused by transfusion of un-irradiated blood to an
Patients present with fever, rash and diarrhoea commencing 1-2
Viable T lymphocytes in the transfused component engraft in the
recipient and react against tissue antigens in the recipient.
Human Leucocyte Antigen (HLA)
Acute GVHD of the skin is characterized by varying
degrees of damage to the epidermal keratinocytes.
Degrees of damage:
White cells from a blood sample are a convenient source
of “ tissue ” that the laboratory can use to determine
individual’s HLA type.
Matching of stem cell donor to a recipient is determined by
comparing their tissue types which can be present on
nearly all tissues in the body.
20-30ml of blood sample and white cells are isolated from
whole blood .
Two methods used:
1. Serological testing: white cells are used.
2. DNA testing: where DNA extracted from white cells is
HLA typing reported as A 3,32, B 7,37, DR 1,15 which will
compare the result of recipient and donor stem cell
- Vacuolization of the basal keratinocytes is present.
- Both basal keratinocyte vacuolization and dyskeratotic
keratinocytes are present.
- Focal clefting of the basal layer is formed.
- The epidermis is totally separated from the underlying
Adverse reaction to blood or platelet
transfusion that occurs when the body
This antibody is directed against human
platelet antigen system.
Thrombocytopenia (platelet counts <10 x
109/L in 80% of cases),
typically 7 to 10 days after a blood
Bleeding from mucous membranes and
the gastrointestinal and urinary tracts is
The immune specificity is against a
platelet-specific antigen yet both
autologous and allogeneic platelets are
Platelet antibodies screening
Method: Flow cytometry
Serum samples are tested against
isolated group O donor platelets
typed for following antigens:
( HPA-1a/b, -2a/b, -3a/b, -4a, 5a/b)
Antibody binding to donor platelets
is detected using fluorescentlabeled polyclonal antibodies
specific for human IgG and IgM
Positive result means plateletreactive antibodies detected
Iron overload in the liver, heart, pancreas, and endocrine glands in the thalassemic
Hemosiderosis that occur due to blood transfusion may occur after transfusion of as few
as 100 units of blood which each unit contain 250mg of iron.
Early symptoms are often vague such as muscle weakness, fatigue and weight loss.
Later skin pigmentation, arthropathy, diabetes, cardiac failure and hepatic dysfunction
Evidence of iron overload with organ dysfunction, may occur after transfusion of 50 to
100 red cell units.
Each unit of red cells contains about 250 mg of iron and the average rate of iron
excretion is only about 1 mg/day.
Hence in chronically transfused patients, the majority of iron can’t be excreted quickly
enough and iron accumulates in the reticuloendothelial system, liver, heart, spleen and
1. Serum iron/ ferritin
This is a blood test that may be done on a regular basis for high risk
Serum ferritin levels increase as the amount of non-transferrin bound iron
(NTBI) increases in the blood.
Blood ferritin levels that are greater than 1,000 mcg/L indicate iron overload.
Healthy men usually have a serum ferritin of 12-300 mcg/L and healthy
2. Liver biopsy
Liver biopsy to check iron concentration.
While this test may give slightly more accurate results than serum ferritin
levels, it requires a fairly invasive procedure that can lead to
complications, such as infection and bleeding.
If the biopsy shows greater than 7 mg iron per gram of liver, the patient is
considered iron overloaded.
WE HAD EXPLAINED ABOUT ALL
GENERAL LAB INVESTIGATION WHEN
THERE IS PRESENCE OF TRANSFUSION
REACTION..SO NOW,,LETS TEST YOUR
UNDERSTANDING BY EXPLORING THIS
EXAMPLE OF CASE STUDY BELOW
Example of case
acute lung injury
A 25 year old female suffered a broken femur in a car accident,
underwent surgery the next day and received 2 units of packed
red blood cells.
Patient was extubated after adequate spontaneous ventilation
was established. Approximately 3 hours after transfusion and 15
minutes after extubation,
-patient’s respiratory rate increased from 12 to 32breaths/minute.
-temperature rose from 36.7 to 38.7 C.
-blood pressure dropped from 120/70 to 101/74.
-Blood oxygen saturation (Spo2) dropped from 100% to 90%.
-chest x-ray showed severe pulmonary edema.
-Patient’s arterial blood gas (ABG) showed hypoxemia With
PaO2 of 60 mmHG.
-Patient’s oxygen saturation was not maintained above 90% with
O2 supplementation and patient was reintubated.
LAB DIAGNOSIS RESULTS
A differential diagnosis of pulmonary/fat embolism,
aspiration pneumonitis, pulmonary edema, fluid overload,
ARDS and TRALI were suspected.
Chest X-ray showed massive pulmonary congestion with
diffuse infiltrates. Urine sample showed hemolysis.
ETT suction showed blood stained secretions.
Supportive measures were taken in the ICU and patient
showed improvement clinically.
By Post-operative day two, chest x-ray became clear and
patient was weaned and extubated.
Laboratory studies at the blood transfusion service
confirmed the diagnosis of TRALI at a later date.
There are two distinct mechanisms have been suggested to
have caused TRALI which are:1)
An antibody-mediated reaction between recipient
granulocytes and anti-granulocyte antibodies from
donors who were sensitized during pregnancy.
An antibody-mediated reaction between recipient
granulocytes and anti-granulocyte antibodies from
donors who were sensitized during previous transfusion.
TRALI is caused most often when anti-HLA class I and antineutrophil antibodies from blood products are passively
transfused to a recipient.
Less frequent is the recipient antibody reacting to the white
blood cells in the transfused blood product.
The subsequent antibody-antigen reaction in the recipient
activates complement, and C5a produced during complement
activation promotes neutrophil aggregation, margination, and
sequestration in pulmonary microvasculature.
The entry of neutrophils into the lung damages and increases
permeability of the pulmonary microvasculature, leading to
pulmonary edema. This reaction is likely to occur in
Another theory suggest accumulation of lipid product resulting
from cell degradation. This pro-inflammatory molecules
accumulate during storage of cellular blood products.
Early diagnosis of TRALI is important in the supportive
treatment measures of these reactions and should be
suspected with symptoms that may include:Dypsnea
fever along with physical findings of bilateral pulmonary
edema, even many hours after the actual transfusions.
Suspicion of TRALI should be reported to the blood
transfusion service so appropriate action can be taken to
prevent future morbidity and mortality in other patients
Treatment of TRALI is largely supportive, and
oxygen supplementation is needed in almost all
cases. Intubations and mechanical ventilation might
be necessary for severe hypoxemia.