Principle of Flow cytometry test - Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of theNADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the polymorphonuclear cells (PNM) of affected patients. This procedure directly labels and stimulates cells in whole blood enabling the diagnosis of patients with CGD or X-linked carriers. The cells are labeled with Dihydrorhodamine123 which is oxidized when the cells are stimulated to undergo the oxidative burst with phorbol myristate acetate.  The oxidized form of Dihydrorhodamine123 is fluorescent. This technique is particularly useful for the diagnosis of CGD in very young infants due to the small volume of blood required.
 
Control- Unstim(Top), Stimulated(Bottom) Twin #1 (AD)Unstim(Top), Stimulated(Bottom) Twin #2 (JD)Unstim(Top), Stimulated(Bottom)
Neutrophil oxidative index (NOI) = 1,614                           NOI=1                              NOI=2 

Interpretation of Results - The patient exhibits virtually no ability to oxidize the dye with an NOI of less than 2 while the healthy control had an NOI of 1,614. Greater than 100 is considered responsive. These results are diagnostic of CGD.Most CGD patients exhibit lymphadenopathy and hepatosplenomegaly and fungal infections are common (particularly Candida and Aspergillus). This patient presented with this clinical presentation.

The detection of carriers is by the detection of a bimodal peak, one peak expressing normal stimulation and one peak expressing the deficient characteristic. This is due to two populations of granulocytes in the carrier ( ie normal and abnormal ). For example, the average NOI for the population of normal granulocytes in the three x-linked carriers was 192, and the average NOI for the population of granulocytes expressing X chromosomes with the CGD mutation was 2.4. The mother of these infants and their three year old sibling were examined by this test (shown below). Mother exhibits this bimodal peak and is a carrier. The sibling, in fact, also had the disease which explains his poor health and past recurrent infections. The sibling had been maintained by frequent antibiotic treatment without coming to a diagnosis.

Control- Unstim(Top), Stimulated(Bottom) Mother Unstim(Top), Stimulated(Bottom)
Control- Unstim(Top), Stimulated(Bottom) Sibling Unstim(Top), Stimulated(Bottom)
Neutrophil oxidative index (NOI) = 930                        NOI=0.9

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CBC
        (Twin #1)
        (Twin #2)
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        (Twin #2)
T,B,NK cell quantitation by Flow Cytometry
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