HAM'S TEST (Acidified Serum Test) And Procedure

HAM'S TEST (Acidified Serum Test) 

HAM'S TEST (Acidified Serum Test)

Principle of HAM'S TEST

HAM'S TEST 

Procedure 

Reagents for  HAM'S TEST

HAM'S TEST (Acidified Serum Test)

In paroxysmal nocturnal haemoglobinuria (PNH), haemolytic anaemia occurs due to the lysis of some red cells in the presence of complement. 

In vitro, these red cells can be detected by exposing them to complement at an acidic pH, using complement in patient's own serum or any other compatible serum.

Principle

The patient's red cells are exposed to complement in his or her own acidified serum and incubated. The amount of lysis produced in this way is compared with that produced by serum heated at 56°C for 20-30 minutes to inactivate the complement Haemolysis does not occur in inactivated serum. 

Specimen

1. Venous blood divided into two parts, one part collected in anticoagulant and the other in a plain tube.

2. Venous blood from a group 'O' individual collected as for the test sample. Reagents

1.0.2 N HCI

2. Sodium chloride, 0.9% 

Technique

1. Place 0.5 ml of serum in a test tube and inactivate at 56°C for 20 minutes.

2. Wash patient's red cells from the anticoagulated blood three times in saline and make a 20 % suspension in saline.

3. Prepare a 20% suspension in the same way from the normal blood sample (group O).

4. Set up four tubes as follows:

Table no.	1	2	3	4
Fresh normal serum	0.5 ml	0.5 ml	-	0.5ml
Heat inactivated serum	-	-	0.5 ml	-
0.2N HCL	-	0.05ml	0.05ml	-
Patient red cells	0.5 ml	0.5 ml	0.05ml	-
Group “o” red cells	-	-	-	0.05ml

Mix well and incubate at 37°C for 1 hour.

5. Mix and centrifuge.

6. Examine for haemolysis. 

Results 

PNH abnormality is detected by strong haemolysis in tube 2, trace or no haemolysis in tube 1, and no haemolysis in tubes 3 and 4. Haemolysis in tubes 3 and 4 shows a false positive test or some rare congenital conditions.