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Compendium Hematopathology > Leukocyte indices and detection of variant hemoglobins > Flashcards

Leukocyte indices and detection of variant hemoglobins Flashcards

1
Q

Leukocyte indices

A
  • RBCs lysed, particles > 36 fL counted as leukocytes
  • light scatter may be used to determine leukocyte differential
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2
Q

Rapid detection of hemoglobin S

A
  • Hemoglobin solubility (dithionate) test
    • detects insoluble forms of hemoglobin within a lysate of blood
    • marked turbidity is a positive screen
    • sensitive to any hemoglobin with altered solubility; may be positive in
      • SS
      • SC
      • SD
      • SA
      • C-Harlem
  • Sickling (metabisulfite) test
    • detects cells that contain sickling hemoglobins
    • smear examined microscopically after metabisulfite is added
    • may be positive in
      • SS
      • SA
      • SC
      • SD
      • C-Harlem
    • requires at least 10% HbS to be positive
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3
Q

Detection of hemoglobin F

  • test methods
A
  • acid elution technique (Kleihauer-Betke)
    • HbA elutes from red cells, but HbF does not
    • cells with persistent eosinophilia contain HbF
    • the heterocellular pattern (some but not all cells contain HbF) is most common pattern
      • typical of fetomaternal hemorrhage and thalassemia
    • pancellular pattern (all cells contain HbF) seen in hereditary persistence of fetal hemoglobin
  • Alkali denaturation technique
    • HbF is resistant to alkali denaturation (in 1.25 M NaOH)
    • HbA is denatured and precipitated out
    • optical density of the remaining supernatant reflects quantity of HbF
  • High pressure liquid chromatography
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4
Q

Hemoglobin electrophoresis

  • sample prep
  • normal values
A
  • lysed blood on cellulose acetate at pH 8.6 (alkaline electrophoresis) subjected to EM force, fixed, and stained
  • normal adult
    • >97% HbA
    • <3% HbA2 (seen in C band)
    • nothing else
  • quantity of Hb variants can be determined by density of gel reading
    • usually not sufficiently accurate for quantification of small quantities of HbA2 or HbF
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5
Q

Hemoglobinopathies in electrophoresis

  • what to do when there is uncertainty on alkaline gel
  • most common fast hemoglobins on alkaline gel; can be mimicked by
  • Hemoglobins in the S region
A
  • Hemoglobinopathies usually produce distinct band on EP
    • when there is uncertainty, an EP on citrate agar at pH 6.2 (acid EP) can help
    • Fast hemoglobins migrate beyond HbA on the alkaline gel
      • can be mimicked by hyperbilirubinemia (esp. in neonates)
      • most common fast hemoglobins are HbH and HbBart
    • When a band is present in the S region, its identity can be confirmed by the sickle screen
      • if screen is negative, this may be D, G, or Lepore
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6
Q

Thalassemia on electrophoresis

A
  • does not usually produce abnormal bands on the EP
    • beta thalassemia diagnosed by presence of thalassemic indices and increased HbA2
    • alpha thalassemia has thalassemic indices and normal HbA2
      • can be mimicked by beta thalassemia with iron deficiency
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7
Q

High pressure liquid chromatography in hemoglobinopathy diagnosis

A
  • Unlike EP, it can
    • separate HbS from HbD, HbG, and Hb-Lepore
    • accurately quantify A2 or F
  • individual molecules elute at different and characteristic rates
  • light source produces a deflection on a spectrophotometer proportional to Hb variant concentration
  • Limitations with HPLC
    • cannot reliably separate HbE and HbA2 (can be accomplished by capillary EP)
    • HbC and HbOArab cannot be easily separated for quantification
    • bilirubin elutes with Hb Barts on HPLC
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8
Q

Molecular methods for hemoglobin identification

A
  • 1-2% of variant hemoglobins detected by HPLC or gel EP cannot be definitively identified
  • sequencing the hemoglobin gene by PCR can characterize the exact genotype for a given individual
  • especially useful in prenatal diagnosis
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9
Q

Pulse oximetry

A
  • 2 wavelengths of light emitting diodes (LEDs) - 660 nm (red) and 940 nm (IF)
    • deoxy Hb has absorption peak at 660 nm
    • oxy Hb = 940 nm
    • can estimate arterial oxygen saturation (SaO2)
  • cannot measure carboxyhemoglobin, methemoglobin, or sulfhemoglobin and will overestimate oxygen saturation in these settings
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10
Q

Arterial blood gas analyzers

A
  • calculate percent saturation after directly measuring the pH, pCO2, and PO2
  • calculation assumes normal Hb-O2 saturation curve, normal 2,3-DPG, and absence of abnormal hemoglobins
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11
Q

Cooximeter

A
  • multiple wavelengths of light
  • can measure
    • oxyHb
    • deoxyHb
    • carboxyHb
    • metHb
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Compendium Hematopathology (8 decks)

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