Haematology Flashcards
What is the difference between acute and chronic Leukaemia?
Leukaemia is a type of cancer that originates in the cells of the bone marrow and results in the overproduction of abnormal white blood cells. The distinction between acute and chronic leukaemia is based primarily on the rate of disease progression and the maturity of the affected cells.
Here’s an overview of acute versus chronic leukaemia:
Acute Leukaemia
Characteristics:
Rapid Progression: Acute leukaemia develops quickly and can progress over days to weeks. Without prompt treatment, it can quickly lead to life-threatening complications.
Immature Cells (Blasts): It involves the accumulation of immature white blood cells, known as blasts, in the bone marrow and blood. These blasts are poorly differentiated, meaning they do not develop into mature, functional cells.
Types:
Acute Myeloid Leukaemia (AML): Affects myeloid cells that form white blood cells (other than lymphocytes), red blood cells, or platelets.
Acute Lymphoblastic Leukaemia (ALL): Affects the lymphoid lineage, which forms lymphocytes.
Clinical Signs and Symptoms:
Symptoms appear suddenly and worsen rapidly. Common symptoms include fatigue, frequent infections, fever, easy bruising or bleeding, and significant anaemia.
Diagnosis involves blood tests showing high numbers of blasts and bone marrow biopsy confirming the presence of greater than 20% blasts in the marrow.
Treatment:
Intensive chemotherapy is typically required soon after diagnosis. This may be followed by additional treatment modalities such as stem cell transplantation, targeted therapy, or radiation therapy, depending on the subtype of leukaemia and patient factors.
Chronic Leukaemia
Characteristics:
Slow Progression: Chronic leukaemia develops slowly, often over months to years, and many patients are asymptomatic at the time of diagnosis.
Mature Cells: Involves more mature forms of white blood cells, which can function nearly normally for some time. The disease gradually results in an excessive accumulation of these relatively mature cells.
Types:
Chronic Myeloid Leukaemia (CML): Primarily affects myeloid cells and is associated with a specific chromosomal translocation known as the Philadelphia chromosome.
Chronic Lymphocytic Leukaemia (CLL): Most commonly affects older adults and involves the lymphoid cells, particularly B lymphocytes.
Clinical Signs and Symptoms:
Often detected on a routine blood test before symptoms appear. When present, symptoms are typically mild at first and include fatigue, weight loss, night sweats, and mildly enlarged lymph nodes.
Diagnosis typically involves blood tests with differential counts and a bone marrow biopsy confirming the presence of increased numbers of mature cells.
Treatment:
Treatment may not be immediately necessary for some patients until symptoms develop or blood counts indicate disease progression. When treatment is required, it often involves targeted therapies, such as tyrosine kinase inhibitors for CML (e.g., imatinib) or monoclonal antibodies for CLL, and less commonly, chemotherapy.
Summary
Acute Leukaemia: Aggressive, rapid onset, affects immature cells, requires immediate intensive treatment.
Chronic Leukaemia: Slower progression, affects more mature cells, can often be monitored for some time before treatment is necessary.
The approach to managing leukaemia—whether acute or chronic—depends heavily on the specific type of leukaemia, the patient’s overall health, age, and the presence of certain genetic mutations. Accurate diagnosis and subtype classification are essential for determining the most effective treatment strategy.
Describe the diagnostic flow cytometry characteristics of AML, ALL, CML, and CLL
Flow cytometry is a critical tool in the diagnosis and classification of leukaemia. It allows for the rapid analysis of multiple parameters of individual cells in a sample, typically blood or bone marrow, by measuring the expression of cell surface and intracellular markers using fluorescently labelled antibodies. This technique is particularly useful for identifying the lineage and maturity of leukemic cells based on their antigen expression profiles. Here’s how flow cytometry characterizes acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), and chronic lymphocytic leukaemia (CLL):
Acute Myeloid Leukaemia (AML)
Flow Cytometry Characteristics:
Myeloid Markers: AML cells typically express myeloid-associated antigens such as CD13, CD33, and CD117 (c-KIT). Myeloperoxidase (MPO) can also be detected intracellularly, which is highly specific for myeloid lineage.
Stem Cell Markers: CD34 and CD117 are commonly expressed on the blasts.
Lack of Lymphoid Markers: AML blasts generally lack lymphoid markers such as CD3 (T cells) and CD19 (B cells).
Variable Expression: CD14 (monocytic marker) and CD64 may be present depending on the subtype of AML.
Acute Lymphoblastic Leukaemia (ALL)
Flow Cytometry Characteristics:
Lymphoid Markers: ALL cells express markers associated with lymphoid cells. B-cell ALL (B-ALL) will commonly express CD19, CD10, CD22, and CD79a. T-cell ALL (T-ALL) will exhibit CD3, CD5, CD7, and other T-cell markers.
Early Differentiation Markers: Common acute leukaemia antigen (CALLA, CD10) is frequently found in B-ALL.
Terminal Deoxynucleotidyl Transferase (TdT): A nuclear protein that is a sensitive marker for lymphoblasts in both B-ALL and T-ALL.
Lack of Myeloid and Monocytic Markers: Typical absence of myeloid markers like CD13 and CD33.
Chronic Myeloid Leukaemia (CML)
Flow Cytometry Characteristics:
Myeloid and Progenitor Cell Markers: CML cells express CD13, CD33, and are positive for CD34, showing the presence of immature myeloid cells.
Philadelphia Chromosome: While not detectable by flow cytometry, most CML cases are characterized by the presence of the Philadelphia chromosome (BCR-ABL1 fusion gene), which can be confirmed through cytogenetic or molecular techniques.
Mature Granulocyte Markers: Expression of markers associated with more mature granulocytes such as CD11b and CD15.
Chronic Lymphocytic Leukaemia (CLL)
Flow Cytometry Characteristics:
B-cell Markers: CLL cells strongly express CD19, CD20 (typically weaker than normal B cells), and CD23.
Specific Markers: CD5 is uniquely expressed on CLL B cells, in conjunction with CD23, which helps differentiate CLL from other B-cell neoplasms which do not typically express CD5.
Light Chain Restriction: Monoclonal kappa or lambda light chain expression is seen, which is useful to establish clonality.
Absence of T-cell Markers: T-cell markers are not expressed, helping to distinguish CLL from T-cell leukaemia’s.
Summary
Flow cytometry offers a rapid, detailed, and highly informative analysis for distinguishing between different types of leukaemia. The expression of specific cell surface and intracellular markers helps in determining the lineage and developmental stage of the leukemic cells, which is crucial for accurate diagnosis, prognosis, and treatment planning.
Describe the role of the Philadelphia chromosome in the pathogenesis of chronic myeloid leukaemia an the role of specific tyrosine kinase inhibitors in treating it.
The Role of the Philadelphia Chromosome in Chronic Myeloid Leukaemia (CML).
The Philadelphia chromosome plays a crucial role in the pathogenesis of Chronic Myeloid Leukaemia (CML). This abnormal chromosome is a result of a reciprocal translocation between chromosomes 9 and 22, specifically t(9;22)(q34;q11). This translocation leads to the fusion of the ABL1 gene on chromosome 9 with the BCR gene on chromosome 22, creating the BCR-ABL1 fusion gene. The product of this fusion gene is the BCR-ABL1 oncoprotein, a constitutively active tyrosine kinase.
Mechanism of Oncogenesis:
Constitutive Tyrosine Kinase Activity: The BCR-ABL1 oncoprotein has much higher tyrosine kinase activity compared to the normal ABL1 protein. This heightened activity leads to the continuous activation of multiple signalling pathways that control cell cycle progression, apoptosis, and cell migration.
Proliferation and Survival: The pathways activated by BCR-ABL1 promote proliferation and survival of leukemic cells, primarily through the PI3K/AKT, RAS/MAPK, and STAT signalling cascades. These pathways collectively inhibit apoptosis, enhance proliferation, and disrupt normal cell adhesion, contributing to the leukemogenesis in CML.
Genomic Instability: BCR-ABL1 also contributes to genomic instability, increasing the likelihood of further genetic alterations that can exacerbate the disease or contribute to resistance against therapies.
Treatment of CML with Tyrosine Kinase Inhibitors (TKIs):
Tyrosine Kinase Inhibitors (TKIs) have transformed the treatment landscape of CML. These drugs specifically target the ATP-binding site of the BCR-ABL1 oncoprotein, inhibiting its kinase activity and thereby blocking the downstream signalling required for leukaemia progression.
First-Generation TKI
Imatinib (Gleevec): The first TKI introduced and a paradigm-shifting approach to CML treatment. Imatinib binds to the inactive conformation of BCR-ABL1, effectively inhibiting its activity. It has significantly improved the prognosis for CML patients, turning a once-fatal disease into a manageable chronic condition for many patients.
Second-Generation TKIs
Dasatinib (Sprycel): It binds both active and inactive forms of BCR-ABL1 and has activity against a broader range of tyrosine kinases, including SRC family kinases. It is effective in patients who develop resistance to or cannot tolerate imatinib.
Nilotinib (Tasigna): Structurally similar to imatinib but designed to be more potent and selective for BCR-ABL1. It is particularly effective against many imatinib-resistant BCR-ABL1 mutations, excluding T315I.
Third-Generation TKI
Ponatinib (Iclusig): Developed to treat CML with the T315I mutation, which is resistant to first- and second-generation TKIs. Ponatinib binds to both active and inactive forms of BCR-ABL1 and inhibits a range of other kinases, providing an option for patients with resistance to earlier TKIs.
Role in Treatment
Induction of Remission: TKIs are highly effective in inducing cytogenetic and molecular remissions in CML patients.
Maintenance Therapy: Continuous treatment with TKIs is often necessary to maintain remission.
Monitoring: The efficacy of TKIs is monitored through regular measurement of the BCR-ABL1 transcript levels in the blood, helping guide treatment decisions such as dosage adjustments or switching drugs in case of resistance.
Conclusion
The discovery of the Philadelphia chromosome and subsequent development of TKIs represent a landmark in the understanding and treatment of CML. These advances highlight the success of targeted therapy based on the genetic abnormalities driving a particular cancer. Continuous research is essential to address resistance mechanisms and improve patient outcomes further.
Outline the morphological classification of the myelodysplastic syndromes and the risk of transformation to acute leukaemia.
Myelodysplastic syndromes (MDS) are a diverse group of clonal hematopoietic disorders characterized by ineffective haematopoiesis leading to blood cell dysplasia’s and varying degrees of bone marrow failure. One of the significant risks associated with MDS is the potential transformation into acute myeloid leukaemia (AML). The morphological classification of MDS, largely based on the French-American-British (FAB) classification and later refined by the World Health Organization (WHO), helps in diagnosing and providing prognostic information, including the risk of progression to AML.
WHO Classification of MDS (Updated Version)
The WHO classification considers cytogenetic and molecular data along with morphology, providing a more detailed framework compared to the older FAB classification. Here’s a summary of the main MDS subtypes according to the
WHO:
MDS with Single Lineage Dysplasia (MDS-SLD):
Dysplasia in one hematopoietic lineage.
<5% blasts in the bone marrow and <1% blasts in the peripheral blood.
Risk of transformation to AML: Low (~5-10% over several years).
MDS with Multilineage Dysplasia (MDS-MLD):
Dysplasia in two or more myeloid lineages.
<5% blasts in the bone marrow and <1% blasts in the peripheral blood.
Risk of transformation to AML: Moderate (10-15% over several years).
MDS with Ring Sideroblasts (MDS-RS):
Divided into MDS-RS with single lineage dysplasia and MDS-RS with multilineage dysplasia based on the presence of ring sideroblasts in the marrow (≥15% of erythroid precursors).
Similar blast criteria to SLD and MLD.
Risk of transformation to AML: Varies, generally low to moderate depending on whether it is associated with single or multilineage dysplasia.
MDS with Excess Blasts (MDS-EB):
Subdivided into MDS-EB-1 and MDS-EB-2 based on the percentage of blasts:
MDS-EB-1: 5-9% blasts in the bone marrow or 2-4% blasts in the peripheral blood.
MDS-EB-2: 10-19% blasts in the bone marrow or 5-19% blasts in the peripheral blood.
Risk of transformation to AML: High (25-30% for MDS-EB-1 and 35-40% for MDS-EB-2).
MDS with Isolated del(5q):
Characterized by a deletion in the long arm of chromosome 5, often with relatively mild clinical symptoms.
<5% blasts in the bone marrow and <1% blasts in the peripheral blood.
Risk of transformation to AML: Low (~10% over several years).
MDS, Unclassifiable (MDS-U):
Includes cases that do not fit into the other categories, often with a single lineage dysplasia and pancytopenia.
Risk of transformation to AML: Variable, generally low unless associated with high-risk cytogenetic abnormalities.
Risk Stratification
The risk of transformation to AML and the overall prognosis in MDS are also evaluated using scoring systems such as the International Prognostic Scoring System (IPSS) and its revision (IPSS-R), which incorporate:
The percentage of bone marrow blasts.
Cytogenetic abnormalities.
The degree of cytopenia’s (affecting red cells, white cells, and platelets).
These scoring systems help in predicting survival and the risk of progression to AML, guiding treatment decisions that can range from supportive care for lower-risk MDS to more aggressive interventions like stem cell transplantation for higher-risk cases.
Conclusion
Understanding the specific subtype of MDS and its associated risk of leukemic transformation is crucial for managing the disease. This requires a comprehensive diagnostic approach including detailed morphological evaluation, cytogenetics, and molecular testing, which collectively inform the therapeutic strategy and prognosis assessment.
Outline the Rai and the Binet classification system for staging chronic lymphocytic leukaemia.
Hypogammaglobulinemia is most common in what leukaemia?
Hypogammaglobulinemia is the most common immune deficiency in chronic lymphocytic leukaemia (CLL)
More common in B-CLL than T-CLL
What does a light chain restriction indicate?
A light chain restriction indicates some degree of clonality.
If there are excessive kappa or lambda chains, it indicates a cell cloning itself excessively.
Explain cll prognosis based on IgVH
Chronic Lymphocytic Leukemia (CLL) is a form of leukemia characterized by the accumulation of functionally incompetent lymphocytes. It is typically a slow-progressing disease and has a highly variable clinical course. One of the significant prognostic markers in CLL is the mutational status of the immunoglobulin heavy chain variable region (IgVH).
IgVH Mutational Status
1. Background:
B-cell Receptor (BCR) Complexity: B cells undergo somatic hypermutation of their B-cell receptor genes, including IgVH, during the germinal centre reaction to increase the affinity of antibodies they produce. CLL cells can be derived from B cells at different stages of maturation, either pre-germinal centre, germinal centre, or post-germinal centre, which affects their IgVH mutation status.
- IgVH Mutation Status:
Mutated IgVH: CLL cells with mutated IgVH genes have undergone somatic hypermutation. This mutation generally indicates that the CLL originated from B cells that have passed through the germinal centre. Patients with mutated IgVH have a more indolent disease course and better prognosis.
Unmutated IgVH: CLL cells with unmutated IgVH genes have not undergone somatic hypermutation, suggesting these cells are derived from B cells that have not passed through the germinal centre. This group tends to have a more aggressive disease and poorer prognosis.
Prognostic Implications - Survival and Progression:
Mutated IgVH: Typically associated with a slower disease progression and longer median survival. Studies have shown that patients with mutated IgVH may have a median survival of over 25 years from the time of diagnosis.
Unmutated IgVH: Associated with a faster progression of the disease and a shorter overall survival, sometimes less than 10 years from diagnosis. - Response to Therapy:
Patients with unmutated IgVH tend to respond differently to certain therapies compared to those with mutated IgVH. They are often less responsive to traditional chemotherapeutic agents but may still respond well to newer targeted therapies, such as BTK inhibitors (e.g., ibrutinib) or BCL-2 inhibitors (e.g., venetoclax).
Testing and Clinical Management
Diagnostic Testing: Assessment of IgVH mutation status is recommended as part of the diagnostic work-up in CLL. This is typically performed using PCR (Polymerase Chain Reaction) followed by sequencing of the IgVH gene.
Treatment Decisions: While IgVH mutation status is an important prognostic tool, treatment decisions in CLL are based on a combination of factors, including clinical stage, patient age, overall health, symptoms, and other biomarkers like CD38, ZAP-70, and TP53 mutation status.
Watchful Waiting: Due to the often indolent nature of CLL, especially in patients with mutated IgVH, a watchful waiting approach may be appropriate in asymptomatic patients until disease progression necessitates therapy.
Conclusion
The IgVH mutation status serves as a critical prognostic marker in CLL, helping predict the disease course and guide management strategies. Understanding this genetic characteristic allows for more personalized treatment approaches, optimizing outcomes for patients with different disease behaviours based on their molecular profile.
List the differential diagnosis of acute, severe anaemia.
Acute, severe anaemia occurs when there is a significant drop in haemoglobin levels, leading to symptoms such as pallor, fatigue, weakness, tachycardia, and shortness of breath. It can develop quickly and may be life-threatening, necessitating urgent evaluation and treatment. The differential diagnosis of acute, severe anaemia is broad, encompassing various aetiologies such as haemorrhage, haemolysis, and inadequate red blood cell production. Here’s a detailed list of potential causes:
- Haemorrhage (Acute Blood Loss)
Gastrointestinal Bleeding: Ulcers, gastritis, oesophageal varices, diverticulosis, colorectal cancer, or angiodysplasia.
Trauma: Accidents or surgical complications leading to major blood loss.
Obstetric/Gynaecological: Heavy menstrual bleeding, postpartum haemorrhage, or ectopic pregnancy. - Haemolytic Anaemias
Autoimmune Haemolytic Anaemia (AIHA): Autoantibodies directed against red blood cells causing their premature destruction.
Microangiopathic Haemolytic Anaemia: Conditions like thrombotic thrombocytopenic purpura (TTP) or haemolytic uremic syndrome (HUS) where red cells are destroyed as they pass through small, damaged vessels.
Infections: Malaria, babesiosis, or bacterial infections like clostridial sepsis that directly invade or produce toxins damaging red blood cells.
Drug-Induced Haemolysis: Due to medications such as dapsone, methyldopa, or some antibiotics.
G6PD Deficiency: An X-linked genetic disorder leading to episodic red blood cell breakdown triggered by infections, certain foods, or drugs.
Mechanical Haemolysis: From cardiac valve prostheses or extracorporeal circuits like in dialysis. - Bone Marrow Suppression or Failure
Aplastic Anaemia: Idiopathic or secondary to drugs, viruses, or toxins leading to pancytopenia.
Infiltrative Diseases: Leukaemia, lymphoma, or metastatic cancer infiltrating the marrow.
Myelodysplastic Syndromes: Clonal bone marrow disorders causing ineffective haematopoiesis and dysplasia.
Megaloblastic Anaemia: Due to deficiency of vitamin B12 or folate causing ineffective red blood cell production. - Anaemia of Chronic Disease
Chronic Inflammatory, Infectious, or Malignant Diseases: Can suppress erythropoiesis through cytokine effects and iron metabolism disruption. - Nutritional Deficiencies
Iron Deficiency Anaemia: Commonly due to chronic blood loss, poor diet, or malabsorption.
Vitamin B12 or Folate Deficiency: Apart from causing megaloblastic anaemia, can impair red blood cell production. - Chronic Renal Failure
Erythropoietin Deficiency: Kidney disease results in reduced erythropoietin production, essential for red blood cell formation.
Diagnostics to Consider
CBC with Differential: To evaluate haemoglobin, haematocrit, MCV, and reticulocyte count.
Peripheral Blood Smear: To look for abnormal red blood cells, signs of haemolysis, or schistocytes.
Iron Studies: Including ferritin, transferrin saturation, and serum iron.
Bone Marrow Biopsy: If marrow infiltration or primary marrow process is suspected.
Coagulation Profile: Especially if bleeding is suspected.
Direct Coombs Test: For suspected autoimmune haemolytic anaemia.
Stool Guaiac Test: For occult gastrointestinal bleeding.
Imaging and Endoscopic Evaluations: Depending on the suspected source of bleeding or disease.
Identifying the underlying cause of acute, severe anaemia is critical for directing appropriate treatment, which may range from blood transfusions and iron supplementation to specific therapies aimed at the underlying disease process.
Classify the haemolytic anaemias and interpret haematological and biochemical investigations in haemolytic anaemia.
Haemolytic anaemias are a diverse group of disorders characterised by the premature destruction of red blood cells (RBCs), leading to a decreased RBC lifespan and subsequent symptoms related to anaemia. These conditions can be classified based on the aetiology of haemolysis, whether it originates intrinsically within the red blood cell or is caused by extrinsic factors affecting the red blood cell from the outside.
Classification of Haemolytic Anaemias
1) Intrinsic Causes (Intracorpuscular):
These are usually hereditary and stem from defects within the red blood cells themselves.
Membrane Defects: Hereditary spherocytosis, hereditary elliptocytosis.
Enzyme Deficiencies: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase deficiency.
Haemoglobinopathies: Sickle cell anaemia, thalassaemia, unstable haemoglobin diseases.
2) Extrinsic Causes (Extracorpuscular):
These causes are usually acquired and involve factors external to the red blood cells.
Immune-Mediated: Autoimmune haemolytic anaemia (warm and cold antibody types), alloimmune haemolytic anaemia (e.g., haemolytic disease of the new-born, transfusion reactions).
Mechanical: Microangiopathic haemolytic anaemia (e.g., disseminated intravascular coagulation, thrombotic thrombocytopenic purpura), mechanical heart valves.
Infections: Malaria, babesiosis, bacterial sepsis.
Chemicals and Toxins: Lead poisoning, venom from snakes or spiders.
Haematological and Biochemical Investigations:
Diagnosis of haemolytic anaemia typically involves a combination of haematological and biochemical tests to evaluate the presence, extent, and cause of haemolysis.
Haematological Tests
Full Blood Count (FBC): Reveals anaemia with decreased haemoglobin and haematocrit levels.
Reticulocytosis (increased reticulocytes) is typically present, indicating increased red blood cell production as a compensatory mechanism.
Peripheral Blood Smear: May show abnormal red blood cell morphologies, such as spherocytes in hereditary spherocytosis or schistocytes (fragmented cells) in microangiopathic haemolytic anaemia.
Direct Antiglobulin Test (Direct Coombs Test): Positive in autoimmune haemolytic anaemia, indicating the presence of antibodies bound to the surface of red blood cells.
Biochemical Tests
Bilirubin: Elevated levels of unconjugated bilirubin due to increased breakdown of haemoglobin.
Lactate Dehydrogenase (LDH): Typically elevated in haemolytic anaemias as it is released from lysed red blood cells.
Haptoglobin: Decreased or undetectable, as it binds to free haemoglobin released from destroyed red cells, which is then removed by the liver.
Urine Haemosiderin: The presence of haemosiderin in urine indicates chronic intravascular haemolysis.
Other Tests
Osmotic Fragility Test: Used in diagnosing hereditary spherocytosis, this test measures red blood cell resistance to haemolysis when exposed to varying concentrations of saline.
G6PD Assay: To detect G6PD deficiency, particularly after an episode of haemolysis.
Haemoglobin Electrophoresis: Used to identify haemoglobinopathies like sickle cell disease and thalassaemia.
Interpretation of Results
In the evaluation of suspected haemolytic anaemia, the combination of clinical features and laboratory findings is crucial:
Increased Reticulocyte Count confirms active red blood cell production as a response to anaemia.
High LDH, Low Haptoglobin, and Elevated Bilirubin support the diagnosis of haemolysis.
Positive Direct Antiglobulin Test points towards an immune-mediated mechanism.
Abnormalities on Blood Smear can suggest specific types of haemolytic anaemia, guiding further targeted investigations.
By using these diagnostic tools, clinicians can not only confirm the presence of haemolytic anaemia but also discern between various aetiologies to tailor appropriate management strategies.
Explain the principles of the direct antiglobulin (coombs) test and list the main clinical conditions that may be associated with a positive result.
The Direct Antiglobulin Test (DAT), also known as the direct Coombs test, is a laboratory procedure used to detect antibodies or complement proteins bound to the surface of red blood cells (RBCs). The test is crucial for diagnosing conditions that involve immune-mediated haemolysis.
Principles of the Direct Antiglobulin Test:
Sample Preparation: A blood sample is taken, and red blood cells are washed to remove unbound plasma proteins and antibodies.
Addition of Coombs Reagent: The washed red blood cells are mixed with the Coombs reagent, which contains anti-human globulin. This reagent can bind to any human immunoglobulins (IgG, IgM) or complement factors (C3d, C4, etc.) that are adhered to the surface of red blood cells.
Agglutination Detection: If the anti-human globulin finds its corresponding antibody or complement factor on the RBCs, it will cause the cells to agglutinate (clump together). This agglutination is visible and can be assessed under a microscope or by automated methods.
Interpretation: Positive agglutination indicates that there are antibodies or complement bound to the RBCs, suggesting an immune-mediated process affecting the red blood cells. A negative test suggests that no such antibodies or complement are bound to the RBCs, although this does not completely rule out other forms of haemolysis or disease.
Clinical Conditions Associated with a Positive DAT:
A positive Direct Antiglobulin Test is indicative of various clinical conditions where immune mechanisms target red blood cells:
Autoimmune Haemolytic Anaemia (AIHA):
Warm Autoimmune Haemolytic Anaemia: Usually caused by IgG antibodies that react at body temperature, affecting the RBCs predominantly in the spleen.
Cold Agglutinin Disease: Typically mediated by IgM antibodies that react optimally at lower temperatures, leading to RBC agglutination and lysis primarily in peripheral blood vessels.
Drug-Induced Immune Haemolytic Anaemia:
Certain drugs can induce the formation of antibodies that either bind directly to the RBCs or induce the immune system to mistakenly target the red blood cells. Common culprits include cephalosporins, penicillin, and some non-steroidal anti-inflammatory drugs (NSAIDs).
Haemolytic Disease of the Newborn (HDN):
This occurs when maternal antibodies, usually IgG, cross the placenta and target foetal RBCs due to blood group incompatibility (e.g., Rh or ABO blood group systems).
Alloimmune Haemolytic Anaemia:
Post-transfusion reactions where the immune system of a transfusion recipient reacts against donor RBC antigens. This can occur if there is an ABO mismatch or other blood group antigen discrepancies between the donor and recipient.
Infections:
Some infections can induce the production of autoantibodies against RBCs or lead to the non-specific binding of antibodies or complement to the red blood cells. Examples include Mycoplasma pneumoniae and Epstein-Barr virus (EBV) infections.
Systemic Lupus Erythematosus (SLE) and other Collagen
Vascular Diseases:
These systemic autoimmune disorders can produce a variety of autoantibodies, including those that react with RBCs.
The DAT is an essential tool in the diagnostic workup for patients suspected of having immune-mediated red blood cell destruction. Its results must be interpreted in the context of the patient’s clinical history, symptoms, and other laboratory findings to guide appropriate management and treatment.
What are the risks associated with red blood cell transfusions?
- Infectious Risks
Transfusion-Transmitted Infections (TTIs): Despite rigorous screening and testing, there remains a small risk of transmitting infections through transfusions. This includes viruses such as hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV), and less commonly, West Nile virus, Zika virus, and parasites like malaria and Chagas disease.
Bacterial Contamination: Typically associated with platelet transfusions due to their storage at room temperature, but RBCs stored in refrigeration can also become contaminated, albeit less frequently. Bacterial contamination can lead to septic reactions in recipients. - Immunologic Risks
Haemolytic Transfusion Reactions:
Acute Haemolytic Reactions: These are severe and potentially life-threatening reactions caused by ABO incompatibility, where the recipient’s immune system rapidly destroys the transfused RBCs.
Delayed Haemolytic Reactions: Occur days to weeks post-transfusion due to anamnestic antibody response against minor blood group antigens not routinely tested before transfusion.
Febrile Non-Haemolytic Transfusion Reactions (FNHTR): The most common transfusion reaction, characterized by fever and chills during or shortly after a transfusion, typically caused by white blood cell antibodies in recipients reacting against donor white cells.
Allergic Reactions: Ranging from mild (urticaria, itching) to severe anaphylactic reactions, possibly due to recipient sensitivity to donor plasma proteins.
Transfusion-Related Acute Lung Injury (TRALI): A serious and potentially fatal complication caused by donor antibodies reacting with the recipient’s leukocytes, leading to acute pulmonary oedema.
Transfusion Associated Graft vs. Host Disease (TA-GVHD): A rare but often fatal condition in which donor lymphocytes engraft in the recipient, mount an immune response, and attack the recipient’s tissues. It primarily occurs in immunocompromised recipients.
- Non-Infectious Non-Immunologic Risks
Iron Overload: Chronic transfusion therapy can lead to an accumulation of iron in the body, potentially causing damage to vital organs like the heart, liver, and endocrine organs.
Volume Overload (TACO - Transfusion-Associated Circulatory Overload): Especially in patients with compromised cardiac function, the additional volume from transfused blood can lead to acute heart failure.
Electrolyte Imbalances: Transfusion can cause shifts in electrolytes, particularly in situations where large volumes are transfused quickly. Potassium released from stored RBCs can cause hyperkalaemia, which may lead to cardiac arrhythmias.
Hypothermia: In massive transfusions, the large volume of cold stored blood can reduce the recipient’s core body temperature, leading to hypothermia.
Risk Management
To mitigate these risks, blood banks employ rigorous testing and processing protocols, including leukoreduction (removal of white cells from blood products), pathogen reduction techniques, and meticulous cross-matching procedures. Clinicians also follow strict guidelines for blood transfusion, including judicious use of transfusions and monitoring during and after the procedure to manage any adverse reactions effectively.
What is the difference between the direct and indirect coombs tests?
Direct Antiglobulin Test (Direct Coombs Test)
Purpose: The DAT is used to determine whether antibodies or complement proteins are bound to the surface of RBCs in vivo. It is a direct test because it examines the patient’s RBCs directly from a sample.
Procedure:
RBC Sample: A sample of the patient’s RBCs is taken.
Washing: The RBCs are washed to remove unbound plasma and proteins.
Coombs Reagent: Antihuman globulin (Coombs reagent) is added directly to the washed RBCs.
Observation of Agglutination: If the antihuman globulin binds to antibodies or complement already attached to the RBCs, it causes the cells to clump together (agglutinate). This indicates a positive test.
Clinical Applications:
Used to diagnose conditions associated with in vivo coating of RBCs with immunoglobulins or complement, such as:
Autoimmune haemolytic anaemia
Haemolytic disease of the new-born
Transfusion reactions
Indirect Antiglobulin Test (Indirect Coombs Test)
Purpose: The IAT is used to detect antibodies present in the patient’s serum (or plasma) but not bound to RBCs. It is indirect because it involves the addition of test RBCs to the patient’s serum, rather than testing the patient’s RBCs directly.
Procedure:
Patient’s Serum: Serum from the patient is obtained.
Addition of Test RBCs: RBCs of known antigenicity (test cells) are added to the serum. These RBCs carry specific antigens that may react with any antibodies in the patient’s serum.
Incubation: The mixture is incubated to allow potential binding of the serum antibodies to the antigens on the test RBCs.
Coombs Reagent: After incubation, the mixture is washed to remove unbound antibodies, and antihuman globulin is added.
Observation of Agglutination: Agglutination indicates that antibodies in the patient’s serum have bound to the test RBCs and are detected by the antihuman globulin, signalling a positive test.
Clinical Applications:
Used primarily in pre-transfusion compatibility testing (crossmatching) to ensure compatibility between donor and recipient blood.
Also used in the detection of antibodies in maternal blood in cases of suspected haemolytic disease of the new-born.
Summary of Differences
Direct Coombs Test focuses on detecting antibodies or complement already attached to the patient’s own RBCs, used to confirm autoimmune or immune-mediated haemolysis.
Indirect Coombs Test is used to detect unbound circulating antibodies in a patient’s serum that could react with RBCs upon transfusion or during foetal-maternal incompatibility.
Both tests are critical tools in diagnosing and managing conditions associated with immune-mediated processes affecting red blood cells, albeit through slightly different approaches and implications.
What molecules in the membrane of erythrocytes are involved in spherocytosis and eliptocytosis?
