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Useful For
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Diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria (PNH) and for screening/monitoring of PNH clone in cases of bone marrow failure syndrome.
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Method name and description
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Immunophenotyping by flow cytometry.
RBC panel: CD235a and CD59
CD235a is used for gating strategy and CD59 is GPI anchored protein. Data analysis is performed to assess, identify and quantify cells lacking expression of GPI anchored proteins (Type III cells), distinguish them from normal RBCs (Type I cells) and recognize cells that are partially deficient (Type II cells) if they are present.
WBC panel is composed of two tubes:
WBCs (Granulocytes): FLAER, CD24, CD45, CD15, CD33.
WBCs (Monocytes): FLAER, CD14, CD45, CD64, CD33.
lineage markers, such as bright CD15 for granulocytes, or bright CD33 or CD64 for monocytes are used and combined with CD45 for accurate cell gating. Data analysis is performed to assess the expression of FLAER/ GPI-Linked antigen: FLAER /CD24 on granulocytes and FLAER/CD14 on monocytes.
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Clinical information
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Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder characterized by nocturnal hemoglobinuria, chronic hemolytic anemia, thrombosis, pancytopenia, and, in some patients with marrow failure disorders as aplastic anemia, MDS,...
PNH is a hematopoietic stem cell disorder that affects erythroid, granulocytic, and megakaryocytic cell lines. The abnormal cells in PNH have been shown to lack glycosylphosphatidylinositol (GPI)-linked proteins in erythroid, granulocytic, megakaryocytic, and, in some instances, lymphoid cells. Mutations in the phosphatidylinositol glycan A gene, PIGA, have been identified consistently in patients with PNH, thus confirming the biological defect in this disorder.
A flow cytometric-based assay can detect the presence or absence of these GPI-linked proteins in granulocytes, monocytes, erythrocytes, and lymphocytes . A partial list of known GPI-linked proteins includes CD14, CD16, CD24, CD55, CD56, CD58, CD59, C8-binding protein, alkaline phosphatase, acetylcholine esterase, and a variety of high frequency human blood antigens. In addition, fluorescent aerolysin (FLAER)- which is an Alexa488-labeled inactive variant of aerolysin-binds directly to the GPI anchor and can be used to evaluate the expression of the GPI linkage on leucocytes.
For cases with clinical suspicion it is recommended to do the test before blood transfusion as transfusion will affect the test results on RBCs.
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Specimen type / Specimen volume / Specimen container
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Whole blood.
Two Peripheral blood samples collected in EDTA tube. 4 ml is needed in each tube.
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Collection instructions / Special Precautions / Timing of collection
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Regular working hours from Sunday to Thursday (7:00 AM-3:00 PM); however, Cutoff time of receiving specimens is 11:30 AM.
Specimen should be received to lab as soon as possible after blood collection and during working hours of the laboratory.
It is recommended to do the test before blood transfusion as transfusion will affect the test results on RBCs.
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Storage and transport instructions
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Specimen Rejection Criteria
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Aged specimen (>24hour).
Frozen/ over-heated specimens.
Clotted Specimens.
Wrong specimen container.
Leaking or contaminated container.
Unlabeled or improperly labeled container.
Mislabeled specimen.
Improperly filled request form.
No specimen types.
Gross contamination of the specimen.
No request/electronic order received.
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Biological reference intervals and clinical decision values
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An interpretive report will be provided.
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Turnaround time / Days and times test performed / Specimen retention time
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2 working days.
Regular working hours from Sunday to Thursday (7:00 AM-3:00 PM); however, Cutoff time of receiving specimens is 11:30 AM.
For requests after working hours and weekends, prior approval from the lab on-call pathologist by the attending consultant is mandatory for arrangements of the testing.
Specimen retention time: 7 Days.
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