Haemolysis Screen

The haemolysis screen combines reticulocyte count, lactate dehydrogenase, haptoglobin and unconjugated bilirubin with the film and the full blood count to detect increased red cell destruction and to separate intravascular from extravascular haemolysis. Free haemoglobin, urinary haemoglobin and haemosiderin are added when intravascular haemolysis is suspected. The direct antiglobulin test is the key next step.

Haemolysis is suggested by anaemia with a raised reticulocyte count, jaundice, dark urine, splenomegaly or gallstones in a young adult.

Indications. Unexplained anaemia with reticulocytosis or macrocytosis, jaundice with a normal liver profile, dark urine, a positive direct antiglobulin test, suspected transfusion reaction, malaria, new prosthetic heart valve, suspected thrombotic microangiopathy, family history of haemolytic anaemia.

Specimen. EDTA blood for the count and reticulocytes. Serum for lactate dehydrogenase, haptoglobin and bilirubin, separated promptly and protected from light. Avoid traumatic or prolonged tourniquet collection, which causes in vitro haemolysis.

Adult reference ranges are given. Local laboratory ranges take precedence.

Reticulocyte count. Marker of marrow response to red cell loss. Range: 25 to 100 x10⁹/L (0.5 to 2.5%). Raised: haemolysis, haemorrhage, treatment response. Lowered: marrow failure, nutritional deficiency, aplastic crisis.

Reticulocytosis is the most consistent sign, though it may be absent early, in marrow suppression, or when antibodies attack erythroid precursors.

Lactate dehydrogenase (LDH). Intracellular enzyme released when red cells lyse. Range: about 120 to 250 U/L, assay specific. Raised: haemolysis (especially intravascular), ineffective erythropoiesis, tissue necrosis, malignancy, liver disease, myocardial injury, lymphoma. Lowered: no clinical significance.

Very high values (several times the upper limit) point to intravascular haemolysis, megaloblastic marrow, or thrombotic microangiopathy.

Haptoglobin. Plasma protein that binds free haemoglobin and is cleared with it. Range: 0.3 to 2.0 g/L (30 to 200 mg/dL). Raised: inflammation, infection, malignancy, biliary obstruction, steroid therapy. Lowered: intravascular and many cases of extravascular haemolysis, ineffective erythropoiesis, liver disease, congenital anhaptoglobinaemia.

An undetectable level with a raised reticulocyte count is highly specific for haemolysis. In inflammation haptoglobin may be falsely normal.

Unconjugated (indirect) bilirubin. Product of haem breakdown. Range: total bilirubin 3 to 17 micromol/L (0.2 to 1.0 mg/dL). Raised: haemolysis, Gilbert syndrome, ineffective erythropoiesis, neonatal jaundice. Lowered: no clinical significance.

Haemolytic jaundice is acholuric, with unconjugated bilirubin, and rarely exceeds 80 to 100 micromol/L in the absence of liver disease.

Plasma free haemoglobin and urinary studies. Free haemoglobin is raised in intravascular haemolysis, normally under about 0.05 g/L, assay specific. Haemoglobinuria gives a positive urine dipstick for blood with no red cells on microscopy. Urinary haemosiderin appears 2 to 3 days after the onset of intravascular haemolysis and persists for weeks.

A positive urinary haemosiderin indicates established or chronic intravascular haemolysis, for example in paroxysmal nocturnal haemoglobinuria.

Typical haemolytic pattern. Raised reticulocytes, raised LDH, raised unconjugated bilirubin, low or absent haptoglobin, with spherocytes, schistocytes or polychromasia on the film.

Intravascular haemolysis. Haemoglobinaemia, haemoglobinuria, haemosiderinuria, undetectable haptoglobin and very high LDH. Causes: ABO-incompatible transfusion, paroxysmal nocturnal haemoglobinuria, G6PD deficiency crisis, thrombotic microangiopathy (TTP, HUS, DIC), mechanical valves, malaria (including blackwater fever), clostridial sepsis, snake venom, some cold type autoimmune haemolysis.

Extravascular haemolysis. Raised bilirubin and LDH with splenomegaly, and less marked haptoglobin depletion. Causes: warm autoimmune haemolytic anaemia, hereditary spherocytosis, sickle cell disease, thalassaemia, unstable haemoglobins, hypersplenism.

Raised LDH and bilirubin with low haptoglobin but low reticulocytes. Ineffective erythropoiesis (megaloblastic anaemia, myelodysplasia), aplastic crisis from parvovirus B19, or marrow infiltration.

Critical values and findings needing immediate action.

• Rapidly falling haemoglobin with dark urine, loin pain or acute kidney injury.
• Suspected incompatible transfusion, with fever, hypotension and haemoglobinuria.
• Schistocytes with thrombocytopenia and neurological or renal features, indicating possible TTP.
• Severe anaemia with absent reticulocytes in chronic haemolysis (aplastic crisis).

Interferences and pitfalls.

• In vitro haemolysis falsely raises LDH, AST and potassium and lowers haptoglobin.
• Haptoglobin is low in liver disease and congenital anhaptoglobinaemia, which is more frequent in some African populations, and raised as an acute phase protein.
• LDH and bilirubin are non-specific and are raised without haemolysis in liver disease, muscle injury and malignancy.
• Gilbert syndrome raises unconjugated bilirubin without haemolysis.
• Reticulocytes can be absent in haemolysis when the marrow is suppressed.
• Recent transfusion confounds reticulocytes, bilirubin and the film.

Special groups.

• Neonates: bilirubin must be interpreted against age in hours, and haemolytic disease of the newborn and G6PD deficiency are leading causes of early jaundice.
• Pregnancy: haemolysis with raised liver enzymes and low platelets suggests HELLP syndrome.
• Older adults: haemolysis may be the first sign of lymphoproliferative disease.

Follow up tests. Direct antiglobulin test, blood film, G6PD assay, haemoglobin electrophoresis, osmotic fragility test, flow cytometry for paroxysmal nocturnal haemoglobinuria, ADAMTS13 activity, cold agglutinin titre, Donath-Landsteiner test, parvovirus B19 PCR, malaria film, urinary haemosiderin, immunoglobulin levels.