TESTING OF RED BLOOD CELLS, GRANULOCYTES &

MONOCYTES BY FLOW CYTOMETRY

Acupath is currently the only New York State private laboratory certified by the New York State Department of Health to perform PNH testing.  High Quality PNH testing at Acupath Laboratories, Inc. was implemented according to the ICCS recommendations published in the 2010 guidelines.

IN THE NEWS

ACUPATH OFFERS “FLAER” REAGENT FOR DIAGNOSING PNH

FOR ANY QUESTIONS ABOUT PNH TESTING AT ACUPATH, PLEASE CONTACT

ZSUZSANNA VEGH-GOYARTS, PH.D.

ASSISTANT DIRECTOR OF FLOW CYTOMETRY

DIRECT CONTACT:   516-394-5633 or 516-394-5572 

EMAIL:  zgoyarts@acupath.com

Diagnostic Background and Clinical Significance:  

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematological disease characterized by hemolytic anemia, cytopenias and life threatening thrombosis. The complement mediated intravascular hemolysis gives rise to various clinical symptoms of the disease.

PNH is a hematopoietic stem cell disorder caused by a somatic mutation in a single gene, PIG-A on the X-chromosome.  The PIG-A protein plays a role in the synthesis of the GPI (glycosylphosphatidylinositol) anchor proteins.  Patients with the defective gene have a partial or complete deficiency of all GPI-linked proteins (e.g. CD55, CD59, CD14, CD16, CD24 etc.) in a clone of hematopoietic cells.

Two of these proteins, CD55 and CD59 have a major role in protecting red blood cells from lysis by the complement system.  These GPI-linked proteins are absent in PNH clones that cause hemolysis in PNH patients.  The absence of the GPI-linked cell surface molecules is detectable by flow cytometry.

Red blood cell markers CD55 and CD59 are the diagnostic target, however testing only the red blood cells may lead to false negative results, since they are sensitive to lysis and may not be detectable. A recent transfusion may also influence the detection of PNH clones in RBCs. Testing of other cell  lineages is recommended such as granulocytes and monocytes (CD24 and CD14).

Recent research lead to a new reagent FLAER (fluorescent aerolysin) that significantly improved the sensitivity and specificity of the PNH testing by flow cytometry.  The aerolysin molecule that specifically binds to all  GPI-linked molecules is now utilized to detect PNH clones on various WBC cell lineages.  By pairing FLAER with antibodies to other GPI-linked proteins and lineage specific markers, a more specific and detailed analysis is possible by flow cytometry.

Reference:

Paroxysmal Nocturnal Hemoglobinuria, PNH, CD55/CD59, FLAER test, hemolysins, Ham test

Clinical Indications:

Hemoglobinuria, hemolysis, thrombosis, elevated LDH, Coombs negative hemolytic anemia, unexplained cytopenias, aplastic anemia, some myelodyspastic syndromes (RA-MDS)

Specimen Requirements:

3-5 ml peripheral blood in Heparin (green top) or EDTA (purple top) tube, kept at room temperature

(18-24 degrees Celcius).  Indicate date and exact time of collection on the requisition and attach a CBC report.

Transport Instructions:

• Guaranteed STAT pickup on all local flow cytometry specimens by Acupath private courier service.

• Non-local Accounts: Ship via FedEx (provided by Acuapth)

Call ahead to notify flow lab about the arrival of the specimen (testing is time sensitive) - 1-888-ACUPATH (228-7284).  Transport the tube(s) at room temperature (18-24 degrees Celcius) to protect from temperature extremes, and deliver to the lab ASAP, but within 24 hours after blood drawing.

Days Performed: 

Monday through Friday

Flow Cytometry Antibody Panel:

RBCs:  CD59 (GlycophorineA for RBC gating)

Granulocytes:  CD24, FLAER (CD45 and CD15 for Granulocyte gating)

Monocytes:  CD14 and FLAER (CD45 and CD33 for Monocyte gating)

Diagnosis:

Reported by a board-certified hematopathologist within 24 hours (or next business day). 

RBCs:

% PNH type III (negative, complete loss), % PNH Type II (intermediate, partial loss) for CD59

Granulocytes:

% PNH type III (negative, complete loss), % PNH Type II (intermediate, partial loss) for CD24 and FLAER

Monocytes:

% PNH type III (negative, complete loss), % PNH Type II (intermediate, partial loss) for CD14 and FLAER

CPT codes:

88184, 88185x10, 88187