SGH 2002 Haem

Question 12

24yo woman, previously well. Presents with lethargy and dark urine. No Hx of recent illness. ?spleen Hb 82, MCV 102, WCC 11.2, plt 355, reticulocytosis, LDH raised. Film shown: retics, microspherocytes, no agglutination, no fragments What would be the next most useful Ix? (a) examine film for Heinz bodies (b) Ham’s test (c) Direct bili (d) DAT (e) Erythrocyte G6PD deficiency

Pt given is clearly haemolysing, so now need to determine the cause.

Heinz bodies  aggregates of denatured hemoglobin  not normally present in red cells  seen when blood smear stained with supravital dye such as crystal violet Most commonly found in: G6PD deficiency following exposure to oxidant compounds thalassemia

G6PD deficiency leads to haemolysis secondary to oxidative stress and thus would expect to see fragments and bite cells. These are not seen, thus (a) and (e) are both incorrect

DIAGNOSTIC APPROACH – Recognizing hemolysis is not difficult in a classic patient with autoimmune hemolytic anemia, who may have all of the following: • New onset of pallor and anemia • Jaundice with increased indirect bilirubin concentration • Splenomegaly • Presence of circulating spherocytic RBCs • Increased reticulocyte percentage • Increased serum lactate dehydrogenase concentration • Reduced (or absent) level of serum haptoglobin

Following this, laboratory findings, including an examination of the peripheral smear, are used to confirm the presence of hemolysis, and, if possible, the underlying cause.

(c) incorrect as indirect bili is elevated, not direct bili Hemolysis may be the first sign of an underlying systemic disorder, such as thrombotic thrombocytopenic purpura, lupus erythematosus, or chronic lymphocytic leukemia, and may require urgent intervention to prevent death or disease-related complications.

Peripheral smear  Spherocytes  Microspherocytes  elliptocytes  Fragmented RBC (schistocytes, helmet cells) - microangiopathic HA  Acanthocytes (spur cells) - liver disease  Blister or "bite" cells - presence of oxidant-induced damage to the red cell and its membrane, with resulting hemolysis  RBCs with inclusions - malaria, babesiosis, and Bartonella infections  Teardrop RBCs with circulating nucleated RBC and early white blood cell forms, indicating the presence of marrow involvement

Serum LDH and haptoglobin  Elevated LDH (released form lyzed red cells)  Reduced haptoglobin (binds to free hemoglobin)

Reticulocyte count The increase in erythropoietin production induced by hemolytic anemia should raise the reticulocyte percentage above 4 to 5 percent

Other tests  Increased indirect bilirubin  Increased mean corpuscular hemoglobin concentration (MCHC), indicating the presence of spherocytes  Positive direct antiglobulin (Coombs') test in autoimmune hemolytic anemia  Tests for cold agglutinins or the Donath-Landsteiner antibody if symptoms are related to exposure to cold

Testing for intravascular hemolysis  plasma hemoglobin concentration  free hemoglobin in the urine supernatant  hemosiderin in tubular cells in the urine no sooner than about seven days after the incident, in order to allow time for hemosiderin-containing tubular cells to be shed into the urine

Ham test (used to diagnose paroxysmal nocturnal haemoglobinuria) In the Ham test, PNH red cells (0.05 mL) are incubated in separate tubes to fresh acidified serum (0.5 mL), unacidified serum, and heated acidified serum. Lysis is determined by optical density of the supernatant fluid after one hour incubation and the addition of 4 mL of 0.15 M NaCl. A positive test is characterized by significant lysis (greater than 1 percent) in acidified serum; lesser degrees of hemolysis may also occur with unacidified serum and the hemolytic properties of the serum are destroyed by heating.

The Ham test is almost never positive in any other disease; the exception is congenital dyserythropoietic anemia type II or HEMPAS (Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum lysis test).

Antibody detection: the direct Coombs' test The autoimmune hemolytic anemias are usually diagnosed by demonstrating an abnormality in a single clinical laboratory test, the direct antiglobulin test first described by Coombs and therefore sometimes called the direct Coombs' test. In this test, the RBCs of the patient are washed free of adherent proteins and reacted with antiserum or monoclonal antibodies prepared against the various immunoglobulins, particularly IgG and a fragment of the third component of complement, C3d. If either or both of these are present on the red cell surface, agglutination or some other endpoint will be detected.

When these tests are accurately and specifically performed, over 99 percent of patients with warm agglutinin AIHA will exhibit a positive result compared to less than 1 percent of the normal population [4]. It is important to know whether IgG or C3 or both are present on the red cells:

• If IgG is present, then the destruction of the red cells is almost certainly due to this antibody. If C3 is also deposited on the RBCs, then the IgG antibody was able to fix complement and complement may play an additional role in the destruction of the cells. If C3 is not present, then the IgG antibody is not able to fix complement or, as with antibodies directed against the Rh complex, the antigens are too far apart for two IgG molecules to be able to activate complement. In these settings, complement plays no role in the destruction of the cells.

• C3 + (IgG-) 1. the antibody is not IgG; this is the case for IgM cold agglutinins and the rare IgA antibodies or 2. the antibody is IgG but so poorly fixed to the RBC surface that it is removed as the cells are washed in preparation for the test. This reduction in affinity may be temperature-related.

A positive Coomb’s test will be diagnostic of autoimmune haemolytic anaemia which is much more common than PNH, so my answer is (d) DAT (coomb’s)