MANUAL DIFFERENTIAL

Principle of flow cytometry test - This test looks for the expression of glycosylphosphatidylinositol (GPI) anchored proteins. In PNH this glycolipid is missing or defective and therefore all surface proteins which are anchored to the membrane by this glycolipid are also missing or reduced. There are many different proteins which are anchored in this fashion on lymphocytes, monocytes, granulocytes, and erythrocytes. Patients with PNH vary considerably in their severity since the defect arises from a somatic mutation of a hematopoietic stem cell. Depending on the extent of this clone and their progeny will determine the severity. Generally, PNH patients fall into four classes of GPI linked antigen expression. (For more information see PNH tutorial)

Patients whos expression of antigen is completely lacking and the fuorescent intensity is similar to the isotype control. These are designated as having Type III cells.
Patients who have a population of cells with totally deficient expression (Type III) and a population of cells with normal expression (designated Type I cells)
Patients who have a poplation of cells with partial (lower flourescence than normal) antigen expression (designated as Type II cells) and a population of normal Type I cells
Patients who have a combination of all types I, II, and III

CD55 and CD59 (GPI linked proteins)
expression on gated granulocytes

Dual Parameter Contour Plot
Healthy Control	Patient

Single Parameter Histograms
	CD59	CD55
Healthy Control
Patient

Interpretation -The patient's flow cytometry studies supports the diagnosis of PNH with type I (normal expression) and type II (partially deficient) cells present. An examination of the granulocyte expression of GPI linked proteins CD55 (decay accelerating factor-disassembles C3/C5 convertase) and CD59 (protectin-binds C8,C9 and prevents assembly of MAC) reveils the classic bimodal peaks. The data has been expressed both as a dual parameter contour plot and as single parameter histograms. The bimodal peaks are easily identified in both presentations of the data.

CD14 (GPI linked protein) and CD45 (non-GPI linked protein)
expression on gated monocytes

Dual Parameter Contour Plot

Healthy Control Patient

Single Parameter Histograms

CD14 CD45

Healthy
Control

Patient

Interpretation -The patient's flow cytometry studies examining the expression of GPI linked CD14 (lipopolysaccharide binding protein) on monocytes is similar to the results found for the GPI linked proteins on granulocytes. The bimodal expression of CD14 is again readily appearant by both presentations of the data compared to the healthy control. Of note, the NON-GPI linked protein CD45 (leukocyte common antigen) is normally expressed by both the patient and the healthy control.

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CBC
WBC with differential
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Bone Marrow results
Other Tests (including Flow Cytometry)

Single Parameter Histograms
	CD14	CD45
Healthy Control
Patient