Fetal Hemoglobin Fetal cells and Fcells by Flow Cytometry

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Fetal Hemoglobin(Fetal cells and
F-cells)byFlow Cytometry
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Utility of Detection of Fetal RBCs

• Detection of fetal red blood cells in maternal
  circulation (Rh incompatibilities) for RhoGAM
  administration
• Percent Fetal rbcs/100 x 5000mls mls Fetal
  rbcs
• One dose Rhogram/30mls Fetal rbcs
• Detection fetal rbcs in acute fetal-maternal
  hemorrhage
• Trauma
• Obstetrical emergencies

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Utility of Detection of Fetal Hemoglobin in Adult
RBCs (F-Cells)

• Detection of F-cells (Adult rbcs which contain a
  portion of cellular hemoglobin as HbF). Normally
  less than 5 of rbc population
• Hereditary Persistence of Fetal Hemoglobin
• Sickle Cell Disease (therapeutic monitoring)
• Thalassemia
• Myelodysplastic syndromes
• Dyserythropoeisis
• Hemolytic anemias

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Kleihauer-Betke (KB) test for detection of Fetal
Hemoglobin in RBCs

• Introduced in 1957
• Acid elution of maternal hemoglobin (fetal
  hemoglobin is acid-stable), stain with
  erythrocin)
• Microscopic examination
• Limited reported sensitivity(0.5 Fetal cells -
  approx. 25mlfetal bleed)
• Inter-observer variability(CV 50-100)
• Time consuming
• F-cells not quantitated accurately
• No longer method of choice BUT still most popular

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Methods Utilized for Detection of Fetal
Erythrocytes CAP 2002
Total laboratories 959

• Kleihauer Betke/Acid Elution 96
• Flow Cytometry 4
• ELISA (ELAT) 0

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Performance of Methods for Detection of Fetal
Erythrocytes - 2002

HBF-01 0.4, HBF-02 0.8 target values
From CAP 2002 participant summary report
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SUNYSB Detection of Fetal Hemoglobin by Flow
Cytometry

• Replaced KB test (2/2001)currently 22/959 labs
  using
• Extremely accurate andsensitive (1/100,000
  cells)
• Linear over large range
• Excellent for detectionof both Fetal rbcs and
  F-cells
• CAP now has proficiencyfor F-cells

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Flow Cytometry Method

• RBCs fixed, permeabilized,washed
• Label with FITC monoclonal Ab to fetal
  hemoglobin,wash
• Resuspend in 1 formaldehyde
• Analyze

Adult cells
Fetal cells
F-cells
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Normal
Normal Range 0.00-0.15
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Fetal BleedTrauma
MVA 36 weeks gestation twins Delivered
only one survived 1.81 91 mls
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HbF and F cells in Thalassemia

• Therapy erthropoietin increases in F cells
  parallels improved clinical course
• Diagnosis recent studies suggest aid in
  distinction between genotype Am J Clin Pathol
  2002 117857-863 (Table below)

Peak Pattern of F-cells
Fl intensity number
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HbF and F cells in Thalassemia
HOMOCELLULAR PATTERNS WITH DIFFERENT HBF CONTENT
MPC Fl 250
MPC Fl 500
HbF 40.5
HbF 81.2
Hereditary persistence of fetal hemoglobin (HPFH)
beta-thalassemia
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HbF and F cells in Thalassemia
HETEROGENEOUS CELLULAR PATTERN
MPC Fl 250
MPC Fl 11
HbF 12.2
delta/beta thalassemia trait
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ThalassemiaIntermedia
42 F-cells
0.02 Fetal cells
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Hereditary Persistence of Fetal Hemoglobin
89 F-cells
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F cells in other diseases

• Sickle Cell
• Therapy - Hydroxyurea increases F cells
• Identification of patients which would most
  benefit
• Therapeutic monitoring

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F- cells in Myelodysplastic syndrome

• Adult MDS (n77) Br J Haematology 93601-605,
  1996
• 62 MDS with ABNK had F-cells gt 5
• 11 MDS with NK had F-cells gt 5
• Level of HbF and F-cell percentages have been
  shown to closely correlate
• CMML in childhood (n110) Blood 893534-3543,
  1997
• Elevated HbF, poor prognosis, short survival
• Pediatric myelodysplasia (n68) Blood
  851742-1750, 1995
• Non-elevation, good prognosis
• More Studies required utilizing Flow techniques
  for accurate measurements

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Summary

• Flow cytometry has replaced K-B Fetal
  hemoglobulin as method of choice
• FC extremely sensitive and accurate
• Majority of labs still using antiquated KB
  method
• F-cell determination may have prognostic and
  diagnostic utility
• MDS, HPFH, Sickle Cell Disease,Thalassemia,
  Dyserythropoeisis, Hemolytic anemias
• Additional studies required, as many early
  studies demonstrating effect used K-B method