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
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
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
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
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
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
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
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
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
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
11
Q
Cooximeter
A
- multiple wavelengths of light
- can measure
- oxyHb
- deoxyHb
- carboxyHb
- metHb
12
Q
A
13
Q
A
14
Q
A
15
Q
A