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Haemolysis is the breakdown of red cells leading to the release of hemoglobin into the surrounding liquid. Haemolysis can be caused by exposure of red cells to complement fixing antibodies, toxins, or mechanically through exposures to broken blood vessels.
Anaemia occurs due red cells being destroyed and removed from circulation before their normal lifespan. Haemolytic anaemia is a form of anaemia due to haemolysis; the abnormal breakdown of red blood cells (RBCs) either in the blood vessels (intravascular haemolysis) or elsewhere in the human body (extravascular). It has numerous possible causes, ranging from relatively harmless to life-threatening.
Classification and causes of haemolytic anaemia:
The general classification of hemolytic anaemia is either inherited or acquired. In inherited haemolytic anaemia, abnormal red cells are made because the gene that controls how red blood cells are made is faulty. This fault can be in the haemoglobin e.g in haemoglobinopathies (thalassaemia and sickle cell disease) (Lucas and National Confidential Enquiry into Patient Outcome and Death., 2008), in red cell membrane as in hereditary spherocytosis or in the enzymes that maintain the integrity of the red cells e.g. G6PD deficiency.
In contrast, in acquired haemolytic anaemia, the body makes normal red cells but environmental factors such as disease, immune disorders, blood transfusion, reactions to drugs or hyperspenism may lead to cell destruction.
Investigation of haemolytic anaemia
Full blood count
The initial laboratory diagnosis of anaemia is always suggested by examination of a Full Blood Count (FBC) result. This may be reflected by a decreased haemoglobin (HB), and usually platelet count.
Although a raised reticulocyte count may be an indication of bone marrow compensation for the haemolysed red cells. This can only be useful if haemolysis has been going for some time. There may be no increase of reticulocyte count if haemolysis is fresh. Besides, any underlying deficiency of haematinics may also lead to low reticulocyte count despite the presence of haemolysis.
Peripheral blood smear microscopy usually shows red cell fragments (schistocytes). In cases where haemolysis is due to hereditary spherocytosis, some red blood cells may appear smaller, rounder and deeply staining than usual. These cells are called spherocytes. Some bluish staining (polichromasia) larger cells may also be present. These are reticulocytes that may be demonstrated using supravital stains. Reticulocytes are present in elevated numbers indicating bone marrow regeneration. This may be overlooked if a special stain is not used.
Bilirubin: During extra-vascular haemolysis, when red cells are phagocytised, haemoglobin is broken down to its component parts (haem and globin). Amino acids from globin chains are returned to the amino acid pool. Iron from haem is returned to (iron stores) in the bone marrow; protoporphyrin ring is converted to bilirubin for ultimate excretion through the intestines where it is converted to urobilinogen. Breakdown of protophorphyrin may lead to an increase of free unconjugated bilirubin which can be measured in serum, as urobilinogen in urine or stercobilinogen in stool (Robert S. Hillman, 2005). Blood lactate dehydrogenase (LDH) may also be elevated.
Haptoglobins: A group of Î±2-globulins in human serum, so called because of their ability to combine with hemoglobin, preventing loss in the urine; variant types form a polymorphic system, with Î±- and Î²-polypeptide chains controlled by separate genetic loci. Levels are decreased in hemolytic disorders and increased in inflammatory conditions or with tissue damage. During haemolysis, the resulting free haemoglobins form complexes with haptoglobin, with the complexes being broken down in the liver. As a result, the reduction of serum haptoglobin is an indication of free haemoglobin which is an indicator of haemolysis. Free haem is oxidised to methaem that is bound by haemopexin for catabolism by the liver or bound by albumin to form methaemalbumin which remains in circulation until more haemopexin is available (Greer, 2006, Robert S. Hillman, 2005).
If the direct Coombs test is positive, hemolysis is caused by an immune process.
Hemosiderin in the urine indicates chronic intravascular hemolysis. There is also urobilinogen in the urine.
The tests for haemolysis identify the different aspects of the haemolytic processes described above. The full blood count, though being the initial indicator of the haemolytic process, is futile without the aid of the other tests such as biochemistry tests. However the study of cytograms has revolutionised the amount of information generated by the initial FBC, as it is now possible to characterise haemolytic disease from the scattergram. The problem, however is that the uptake, or understanding of the information generated on scattergrams is still very low among laboratory practitioners.
The interpretation of blood films to exclude other causes of haemolysis may result in a delay of a diagnosis of haemolytic disease being made as a conclusive diagnosis may require confirmation by a haematology consultant. However, other aspects of the blood film, not commonly used by Biomedical Scientists, present themselves on some analysers, for example a disparity between cellular and plasma haemoglobin, where the plasma haemoglobin increases. An increase in plasma haemoglobin may therefore imply intravascular haemolysis, and may, for example, indicate immediate transfusion reaction. A reduced platelet count may also result from increased lytic action of complement.
The size of cells may suggest what type of haemolysis is occurring, for example if the anaemia is due to membrane defect, normal size cells are formed. On the evidence of a low haemoglobin and raised bilirubin, the doctors may suggest a reticulocyte count. The reticulocyte count may suggest haemolysis as the bone marrow compensates for the anaemia by releasing immature red cells.
As indicated above, the most crucial investigation in the diagnosis of haemolytic anaemia is performance of a peripheral blood smear, on which spherocytes and cell fragments (schistocytes) may be seen. The blood smear review may reveal nucleated red cells, polychromasia (reticulocytes), and spherocytes.
The performance of a direct antiglobulin test (DAT), may, if positive be suggestive of immunologic basis for haemolysis due to sensitisation of the red cells. A negative DAT may not exclude haemolytic disease as Paroxysmal Nocturnal Haemoglobinuria (PNH) is not antibody mediated.
Biochemistry tests can also be performed such as haptoglobin, where a decrease of haptoglobin may be due to increase in plasma HB/Haptoglobin complexes which are cleared by the reticulo-endothelial system, resulting in reduced free haptoglobin. Other changes which may be used includes, the presence of free haemoglobin in urine, this is because haemoglobin from intravascular haemolysis is cleared by the kidney. Therefore the haemoglobin appears in urine.
Other changes which can be investigated when haemolysis occurs include urine haemosiderin, which when positive indicates chronic haemolysis, this is because when chronic haemolysis occurs, iron is lost in urine. Renal tubular cells catabolise iron, and when the tubular cells are sloughed into urine, the Prussian blue stain reveals iron in urine. Less used tests, and possibly too invasive for this purpose due to the discomfort to the patient is bone marrow aspiration, however this has the potential to demonstrate the compensatory production of immature red cells , many of which get released prematurely into circulation. The Donath-Landsteiner test is used to demonstrate the non-immunologic destruction of red cells in PNH.
The Heinz body anaemias occur when enzyme deficiency results in loss of the reduction mechanism of the oxidised haemoglobin molecules, this can be investigated by performance of the fluorescence spot assays which demonstrate deficiency of G6PD or PK. These are useful tests, however as haemolysis progresses, the reticulocyte count increases, and reticulocytes contain high quantities of G6PD/PK. This results in the spot assays appearing like there is a normal haemoglobin concentration. Therefore the performance of these assays must be combined with performance of the reticulocyte count, and the test not performed when the reticulocyte count is high or the test interpreted with caution. Other assays which may be used in this case would be the methaemoglobin assay, which when elevated would reflect the haemolytic process, and is less affected by changes in the reticulocyte count.
The chronic anaemias leading to haemolysis may be investigated leading on from the blood film findings suggestive of thalassaemias or haemoglobinopathies can be followed by haemoglobin electrophoresis; then confirmation can be done to identify the specific variant. If the Haemoglobin electrophoresis results are normal then tests for the sensitivity of the red cell membranes to lysis can be done. These include the osmotic fragility tests, and the acidified serum lysis tests. However these tests are becoming obsolete as new ways of investigation become more popular.
The diagnostic methods described above are not exhaustive, and are done in consultation with the clinical history, and ethnicity of the patients. Molecular methods may be used to identify certain haemoglobin variants.
In conclusion, the diagnosis of a haemolytic anaemia must address the pattern of changes that are likely to occur during a haemolytic process. While some of these areas may be the cause of the haemolysis, others may be due to by-products of haemolysis or indications of increased bone marrow activity. These could include factors like; the red cell membrane is more sensitive to lysis than usual due to membrane abnormalities leading to reduced red cell deformability. Underlying enzyme deficiency may lead to a compromised red cell lifespan when subjected to oxidative stress. Other hereditary abnormalities like haemoglobinopathies e.g. thalassaemias, sickle cell anaemia and other variant haemoglobins may lead to cell being removed prematurely from circulation.
Red cells coated with antibodies may be removed from circulation by the cells of reticuloendothelial system (RES). The other mechanism of red cell destruction can be through attachment of immunoglobulin to the cell membrane followed by complement fixation ending in red cell haemolysis.
As a result haemolysis, there is a change in storage and utilisation of iron, and there is a response to the anaemia by the bone marrow which is dependant on the potential of the bone marrow. There is also increased elimination of waste products of haemolysis from urine and stool.
GREER, J. P. (2006) Wintrobe's Clinical Hematology. Book, 1, 160.
LUCAS, S. B. & NATIONAL CONFIDENTIAL ENQUIRY INTO PATIENT OUTCOME AND DEATH. (2008) A sickle crisis? : a report of the National Confidential Enquiry into Patient Outcome and Death (2008), London, National Confidential Enquiry into Patient Outcome and Death.
ROBERT S. HILLMAN, K. A. A., HENRY M. RINDER (2005) Hematology in clinical practice: a guide to diagnosis and management. 136-139.