Hemolytic Transfusion Reactions (ABO incompatibility)

I. What every physician needs to know.


Blood group antigens on red blood cell (RBC) surfaces define their immune potential. The different blood groups A, B, AB and O are based on the surface presence of antigen A, antigen B, both antigens or absence of these antigens, respectively. Corresponding to these blood groups, IgM antibodies (anti-A, anti-B) are found in the plasma of adults lacking the corresponding antigen. Hence, blood group A, B, O, and AB have anti-B, anti-A, both, or none of the isoagglutinins (Rule of Landsteiner). Apart from these, there are ‘minor’ alloantibodies such as anti-D, anti-K, and anti-Jka which are present in varying proportions in the population.

Transfusion of blood components can cause a multitude of reactions, not all of which are immune-mediated and not all of which lead to hemolysis (see “Transfusion reactions”). Transfusion entails the merging of two antigenically charged pools of blood components – donors with recipients – thus pre-transfusion compatibility testing is essential to minimize interactions.

Compatibility testing pre-transfusion involves blood typing and cross-match (via the indirect antiglobulin test – see Coombs test below) to ensure the recipients’ blood lacks antibodies that can react with donor antigens and lead to destruction of transfused cells. In spite of compatibility testing, a conglomeration of system and process errors may still lead to transfusion of mismatched blood leading to incompatibility reactions, generally as a clerical or procedural error.

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Key definitions
  • Hemolytic transfusion reactions (HTRs): The occurrence of a serologic reaction (acute reaction) or alloimmunization (delayed reaction) as a result of blood component therapy which leads to a clinically relevant acute or delayed decrease in RBC survival.

  • Delayed serologic transfusion reactions (DSTRs): The occurrence of alloimmunization as a result of blood component therapy without any associated hemolytic anemia.

Types of hemolytic transfusion reactions

Immune mediated hemolytic transfusion reactions

These can be acute or delayed in onset:

  • Acute HTRs: These are due to preformed antibodies against donor RBC antigens present in the recipient’s blood. ABO incompatibility reactions are the most dreaded hemolytic transfusion reactions due to their ability to cause intravascular hemolysis. Non- ABO incompatibility reactions due to minor recipient antibodies (anti-Rh, anti-Kidd or anti-Jka) tend to be milder and generally lead to extravascular hemolysis of the antibody coated RBCs. However, occasionally high titers of these antibodies may involve complement fixation and intravascular HTRs.

  • Delayed HTRs: These are due to an anamnestic response to donor RBC antigens which produces antibodies after a lag period of 3-10 days. This requires prior sensitization in the form of pregnancy, transplantation or transfusions.

Non-immune-mediated hemolytic transfusion reactions

These are generally related to improper storage and handling of blood leading to hemolysis in vitro prior or during transfusion:

  • Thermal injury: During re-warming of the blood if temperatures reach more than 42°C

  • Cold injury: Inappropriate storage with exposure to ice or temperatures less than 6°C

  • Mechanical injury: Lysis during transfusion through small-bore catheters.

  • Infection

  • Concomitant drug-induced hemolysis

  • Concomitant administration of hypotonic solutions (D5%W, hypotonic saline) leading to osmotic injury

Pathophysiology of ABO incompatibility HTRs

The A and B antigens are the most immunogenic; hence transfusion of an ABO incompatible unit causes the recipient antibodies to interact with the donor RBC surface antigens, triggering complement activation and resulting in the acute intravascular hemolysis of the transfused donor RBCs.

Complement activation causes various pro-inflammatory effects via release of active C3a and C5a subcomponents. These are anaphylatoxins which cause histamine and serotonin release from mast cells, increase vascular permeability causing a capillary leak syndrome and stimulate smooth muscle contraction. These translate clinically into the classical symptoms of flushing, hypotension and bronchospasm, respectively.

Experimental evidence supports a central role for cytokines in the pathophysiology of hemolytic transfusion reactions. Tumor necrosis factor (TNF) appears to be the most commonly identified mediator of intravascular coagulation and end-organ injury although other cytokines have been implicated including interleukin (IL)-8, monocyte chemoattractant protein, and IL-1 receptor antagonist.

Ultimately, the complement cascade terminates with activation of the membrane attack complex (MAC) leading to cytolysis. The released hemoglobin tetramers complex with haptoglobin and are removed by the liver, causing haptoglobin depletion. Residual free hemoglobin circulates in the plasma or gets converted to oxidized methemoglobin in the blood, imparting a reddish or brownish color, respectively.

Small, unbound hemoglobin dimers are also filtered by the glomerulus which causes hemoglobinuria. This heme pigment causes acute kidney injury directly or via tubular obstruction or vasoconstriction. In practice, this heme-induced acute tubular necrosis requires a secondary factor like dehydration, nephrotoxin use or sepsis to translate into significant renal insufficiency.

The hemoglobin is taken up by the renal tubular cells, degraded and the iron is stored as hemosiderin. When these renal tubular cells are sloughed in the urine 3-10 days later, hemosiderinuria becomes detectable.

Tissue factor is expressed on monocytes and endothelial cells and can precipitate disseminated intravascular coagulation (DIC). The exposure of RBC stroma and cytokine activation may also feed the consumptive coagulopathy. In rare cases, the above events may culminate into multi-organ failure.

II. Diagnostic Confirmation: Are you sure your patient has ABO incompatibility?

The following criteria determine occurrence of an ABO incompatibility reaction:

1) Recent or ongoing transfusion of blood products.

2) Acute onset of fever or hemodynamic instability.

3) Evidence of hemolysis on visual examination of blood samples and urine.

4) Evidence of hemolysis on laboratory investigations.

5) A positive Coombs test on recipients’ blood.

A. History Part I: Pattern Recognition:

The symptoms of acute hemolytic transfusion reactions can be initially nonspecific and difficult to differentiate from other transfusion reactions. However, always rule out ABO incompatibility, especially if the reaction is severe. Serious transfusion reactions can develop within minutes of starting a transfusion; therefore, monitoring vital signs on initiation and within the first 15 minutes of a transfusion is essential.

Symptoms due to the serologic reaction and complement activation

Fevers are the most common presenting symptom and close vigilance can abort a transfusion before severe damage is done. ABO incompatibility reactions resemble any other drug reaction initially with malaise, dizziness, chills, backache and anxiety.

Vital signs may reflect systemic inflammatory response syndrome (SIRS) criteria with tachycardia and tachypnea. The anaphylatoxins cause bronchospasm with dyspnea, hypotension, flushing, chest pain, and nausea.

Symptoms of complications due to hemolysis

Since HTRs cause hemolysis of the small volume of donor cells, the main threat is not that of low hemoglobin but that of pigment-induced injury due to the intravascular hemolysis. Depending on the extent of intravascular hemolysis, one could see rapid development of hemoglobinuria and patients may complain of a darkening of urine. Dark urine may be the first indicator of intravascular hemolysis, especially in anaesthetized or unconscious patients. Oliguria is noted on subsequent monitoring as acute renal failure sets in.

Patients with severe ABO incompatibility may develop DIC and show all the symptoms of this disease, including diffuse bleeding. Fatality is rarely seen in modern medicine but would be due to a multiorgan failure following circulatory shock.

In contrast to ABO incompatibility, DHTRs cause extravascular hemolysis and are generally asymptomatic and rarely fatal. Fever and chills are common a week or two after initial blood transfusion and are associated with a decreasing hemoglobin and spherocytosis. Hyperbilirubinemia may be seen but discoloration of the urine is rare due to extravascular hemolysis. Due to the nonspecific signs and symptoms, DHTRs tend to go unrecognized.

B. History Part 2: Prevalence:

Approximately one of every 25,000 transfusions is complicated by ABO incompatibility. Blood group O and A are the most prevalent in the community, accounting for 43% and 44% of the population. Based on this, the most common mismatches are due to transfusion of type A blood to type O individuals who carry both anti-A and anti-B. The reverse scenario, i.e., transfusion of type O blood to type A blood group individuals, is acceptable since group O is antigenically silent.

Any patient receiving a transfusion, irrespective of type and cross-match, remains at risk for incompatibility reactions since most of these are due to clerical or procedural errors, i.e., wrong blood to wrong patient. However certain patients may be particularly at risk.

Specific at-risk patient populations
  • Emergencies where O group blood is transfused while a concurrent blood cross-match is being performed in the laboratory.

  • Short supply of the patient blood type.

  • Solid organ transplantation.

  • ABO mismatched platelet transfusion.

Age distribution of ABO incompatibility reactions
  • Newborns develop antibodies to RBC antigens A and B after 3-4 months of life and hence life threatening ABO-related transfusion reactions are not observed in this age group.

  • At the other extreme there is a steady decline in the titers of ABO antibodies after age 60 which decreases the severity of these reactions. However, most blood transfusions are administered to persons aged 60 years and older; therefore, most acute transfusion reactions also occur in this age group.

C. History Part 3: Competing diagnoses that can mimic ABO incompatibility

  • Any transfusion reaction, including febrile non-hemolytic transfusion reactions and allergic reactions, should be considered in the early differential. They should be differentiated from each other only when laboratory data is available and as the clinical course evolves.

  • Concurrent drugs or inherent patient conditions which may precipitate hemolysis.

  • In trauma patients receiving blood transfusions it may not be uncommon to see hemodynamic instability, acute renal failure and discolored heme-positive urine from rhabdomyolysis. Also, in patients with trauma who are being transfused blood, it may become necessary to distinguish between myoglobinuria and hemoglobinuria both of which can cause urine color changes and a heme-positive dipstick result.

D. Physical Examination Findings.

HTRs may not reveal any specific examination findings. Patients may have fever, rigors, and hemodynamic instability. Jaundice may be apparent. Pulmonary auscultation may reveal wheezing.

E. What diagnostic tests should be performed?

1. What laboratory studies (if any) should be ordered to help establish the diagnosis? How should the results be interpreted?

Transfusion reactions are acute, varied in etiology, and require urgent treatment. This establishes a diagnostic urgency for the practicing physician and requires collaboration with the blood bank and pathologists. However, it needs to be stressed that treatment should be initiated at the earliest without awaiting the final diagnosis.

Steps to diagnosis

The following steps should be undertaken to arrive at the diagnosis:

1) Examining the blood product bag

Since most of the HTRs are due to clerical/process errors, verifying the blood bag identity with the patient identifiers reveals the incompatibility in most cases.

2) Screening tests

  • Complete blood count with peripheral smear

  • Comprehensive metabolic panel

  • Urinalysis

  • Hemolysis panel (haptoglobin and LDH)

3) Confirmatory tests

The direct Coombs test is the diagnostic test for ABO incompatibility reactions.

4) Evaluate competing diagnosis

Though the temporal relationship of the blood transfusion is a good surrogate for an etiologic cause, concurrent drugs and inherent patient conditions may also induce hemolysis in a few patients. A comprehensive evaluation will ensure that alternative etiologies are not missed. (See destructive anemias)

Overview of laboratory tests

1) Complete blood count

Since the volume of the lysed donor PRBCs is small, HTRs do not cause a significant drop in the hemoglobin. Occasionally, a non-significant increase in the hemoglobin post-transfusion can hint towards a HTR with the right clinical picture. Platelet counts and coagulation abnormalities become evident if DIC sets in.

A peripheral blood smear can detect schistocytes and shift cells which are large polychromatic RBCs, suggestive of early release of reticulocytes into the circulation due to erythropoietin stimulation.

2) Comprehensive metabolic panel

An elevation in the unconjugated bilirubin may indicate hemolysis. Hyperkalemia may develop from cell lysis. A basic metabolic panel also provides a baseline creatinine to help monitor for the onset of acute renal failure.

3) Urinalysis

Urinalysis may show hemoglobinuria. Normally hemoglobin does not undergo glomerular filtration due to its large molecular size; however, during high plasma concentrations due to ongoing hemolysis it can be transiently detected in the urine. Depending on the concentration and its state of oxidation, the urine color changes from pink-red to a dark brown, while severe cases of intravascular hemolysis can classically show very dark urine termed “blackwater”.

Degraded hemoglobin gets deposited into the tubular cells as hemosiderin. After 3-7 days, as the tubular cells slough off, hemosiderinuria can be detected by iron staining of the urine.

4) Other tests indicating presence of acute intravascular hemolysis

Elevated LDH and decreased haptoglobin can correlate with the degree of hemolysis. Note that haptoglobin is also an acute phase reactant and may be relatively elevated by a concomitant inflammatory condition.

Hemoglobinemia imparts a pinkish color to the plasma. This requires high levels of free hemoglobin and may be masked by concurrent bilirubin. On automated cell counters it causes a rise in mean corpuscular hemoglobin concentration (MCHC). RBC lysis during venipuncture can also show hemoglobinemia in the blood sample.

The above tests for hemolysis may be less reliable in patients with concomitant liver disease who also have low haptoglobin levels, high bilirubin and high LDH. However serial measurements of these may indicate an active hemolysis.

5) Definitive diagnostic test for incompatibility

Detection of isoagglutinins attached to the RBC surface by the direct Coombs test is the best diagnostic test. However, it can be time-consuming in the setting of an obvious emergent clinical situation and treatment should be initiated at the earliest.

A repeat cross-match with the donor blood should be performed with the indirect Coombs test which detects if potential antibodies against donor RBCs were present in the patient’s serum prior to transfusion.

Coombs test (Anti-globulin test)

There are two types of Coombs test – direct and indirect. The direct Coombs test is the diagnostic test for ABO incompatibility reactions. The indirect Coombs test is used for the pretransfusion cross-match or “screen” to detect the presence of antibodies in the recipient prior to a transfusion.

Direct Coombs test detects if antibodies in the blood have coated the donor RBCs. When antibodies to the human globulin fractions (antiglobulin or Coombs serum) are added to the patient’s blood, they will bind to the surface antibodies and complement particles and cause the donor RBCs to agglutinate. The results are quantified on a scale from 1+ to 4+ indicating a positive test. Absence of agglutination or clumping rules out an antibody-mediated hemolysis. If there has been widespread hemolysis of the donor RBCs, the direct Coombs may be negative. In this case, the precipitous decline in the antibody titres from pre-transfusion levels can help in diagnosis.

III. Default Management

A. Immediate management

If there is a likelihood of an ABO incompatibility reaction, treatment should be initiated while awaiting laboratory data.

In general, the following steps should be followed:

1) The severity of HTRs is generally related to the volume of blood transfused, hence the most important thing is to stop the transfusion and immediately alert the blood bank.

2) Generally, all blood banks have a protocol in place to further evaluate potential HTRs which should be activated and strictly adhered to. A mismatched unit on your patient suggests that there may be another patient at risk for a mismatched unit.

3) Hemodynamic stability should be the prime concern along with ensuring an adequate airway and intravenous access. Patient should be moved to a telemetry monitored unit.

4) Treatment starts with aggressive fluid replacement with normal saline to prevent renal failure. Ringers lactate should be avoided to trigger calcium-induced clotting of the blood in the tubing. Similarly, dextrose solutions can worsen hemolysis of blood in the tubing. The initial rate could be 100ml/hr to 150ml/hr with intermittent fluid boluses to maintain a brisk urine output and avert renal failure.

5) The intravenous tubing, transfusion records (if on paper), donor blood bag should be preserved and sent down to the blood bank.

6) A fresh sample of the patient’s blood from the opposite arm should be sent to the blood bank and blood and urine laboratory investigations should be ordered.

7) Complications should be anticipated, monitored for and treated promptly:

  • Widespread hemolysis can induce circulatory shock which may require vasopressor support.

  • The release of large amounts of potassium and other electrolytes can cause cardiac arrhythmias and may require urgent hemodialysis.

  • DIC should be treated supportively. See DIC

8) If the patient develops complications of acute renal failure, DIC or multi-organ failure, transfer to a higher level of care or intensive care unit may be appropriate.

9) There are some case reports of treatment of ABO incompatibility with eculizumab, a potent C5 inhibitory antibody. This may be considered in consultation with a Hematologist.

C. Laboratory Tests to Monitor Response To, and Adjustments in, Management

Monitoring of vital signs every 4-6 hours with a daily creatinine and hemoglobin may be sufficient in hemodynamically stable patients. Hemolytic data like LDH may help chart the course of the reaction; however, these tests are not routinely needed. Patients should be maintained on telemetry until the risk of acute renal failure and electrolyte abnormalities dissipates.

D. Long-term management

Preventing hemolytic transfusions reactions

HTRs can also happen due to any systematic error along the transfusion chain, from initial request for blood to actual transfusion. Generally, the actual type and screen process is not faulty. Subsequent human and process errors can occur around the two main events – specimen collection from recipient and administration of blood.

The two most common reasons for HTRs due to incompatibility are:

  • Mislabeling of the recipient blood on initial collection.

  • Transfusing blood to the wrong patient.

To target the first shortcoming, a common practice followed by many blood banks is to perform a concurrent blood typing from the other arm for first time transfusion recipients to ensure correct identification of blood type and minimize unrealized system errors.

Once the appropriately crossmatched blood is released, labelled with intended recipient information, stringent patient identification and matching via 2 separate indicators (medical record number, birth date, etc.,) will ensure that there is no mismatch. Two practitioners and if possible, the patient, should verify the match with the information printed on the blood bag.

Early detection of hemolytic transfusion reactions

Repeated studies have found no merit in using pre-transfusion prophylactic antipyretics. In fact, they can mask early constitutional symptoms of a reaction and delay life-saving treatment. Hence, these should be avoided.

Similarly, in non-emergent settings, it is essential to ensure that the patient is afebrile prior to transfusion to avoid confusing this with fever from a transfusion reaction. Also, as mentioned before, since most reactions occur within the first 15 minutes, patients’ vitals should be closely monitored during this time and patients should be informed to report any new symptoms.

Post-hemolytic transfusion reaction analysis

If a patient develops a HTR, use of a sentinel error reporting system (SERS) and ensuring a root cause analysis is performed to decipher the weakest links in the transfusion chain is essential.

In cases of DHTRs, work-up should confirm the antibody formation and provide patients with a list of detectable antibodies to prevent HTRs in the future via transfusions at other medical centers.

IV. Special situations

Hyperhemolytic transfusion syndrome

Hyperhemolysis syndrome is defined as destruction of transfused as well as autologous red blood cells, leading to a precipitous drop in hemoglobin to below pre-transfusion levels. Patients with sickle cell disease present a unique sub-population at risk for hemolytic transfusion reactions due to the following reasons:

  • They tend to have repeated exposure to blood transfusions, resulting in high titers of alloantibodies.

  • There is a discrepancy in the United States between RBC phenotypes of donors (primarily non-African American) and sickle cell recipients (primarily African American), increasing risk of alloimmunization.

This syndrome is rarely seen in patients without underlying hemoglobinopathy. Sickle cell patients are at risk of developing the hyperhemolytic transfusion syndrome which involves a post-transfusion accelerated drop in hemoglobin with a sickle crisis exacerbation due to:

  • Hemolysis of donor RBCs.

  • Hemolysis of autologous RBCs due to high titers of alloantibodies with complement activation.

  • Suppression of hematopoiesis due to the transfusion itself.

These patients can be challenging to manage since additional transfusion may be required to treat anemia but carries the risk of fueling the immune reaction. A prompt Hematology consultation is appropriate. In a few case reports further transfusions have been avoided with the use of corticosteroids and intravenous immunoglobulin (IVIG) or treatment with eculizumab.

ABO mismatched platelet transfusions

Due to limited availability of platelet products they are generally transfused across the ABO barriers. Hence, single donor platelets which tend to have 200-400 ml of plasma can rarely cause ABO-incompatibility hemolytic reactions. Awareness of this possibility is key since the management remains the same.

V. Transitions of Care

D. Arranging for Clinic Follow-up

The patient should be provided with contact information for the hospital’s blood bank. In the case of minor incompatibility, once the complete antibody screens are available it is essential to inform patients about the new antibodies detected. They should be instructed to provide this list when they undergo future transfusions at a different center.

VI. Patient Safety and Quality Measures

A. Core Indicator Standards and Documentation.

Ensuring proper patient identification prior to blood transfusions by matching the blood product to the patient is listed as a Joint Commission National Patient Safety Goal for 2016.

“Updated National Patient Safety Goals”.

Weinstock, C, Möhle, R, Dorn, C, Weisel, K. “Successful use of eculizumab for treatment of an acute hemolytic reaction after ABO-incompatible red blood cell transfusion”. Transfusion. vol. 55. 2015. pp. 605-610. (This article gives a case report of successful treatment with eculizumab to prevent C5 activation and extend the lifetime of transfused incompatible cells.)

Boonyasampant, M, Weitz, I C, Kay, B, Boonchalermvichian, C. “Life-threatening delayed hyperhemolytic transfusion reaction in a patient with sickle cell disease: effective treatment with eculizumab followed by rituximab”. Transfusion. vol. 55. 2015. pp. 2398-2403.

Eberly, L, Osman, D, Collins, N.. “Hyperhemolysis Syndrome without Underlying Hematologic Disease”. Case Reports in Hematology. 8 February 2015.