Page 698 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

682 PART 8 ■ Fundamentals of Hematological Analysis

SPECIAL HEMATOLOGY PROCEDURES (continued)

FIGURE 32.7 Electrophoresis. A. Specimen rom a

neonate. Hb-Barts is present (arrow) in icating a thalas-

semia. B. Specimen rom a 3-month-ol . In a ition to
Hb-Barts, there is also a small amount o Hb H (double

arrow). C. Hb H-Hb-Constant Spring. T ere is a ull-

ness in the Hb A2 area (arrow) in icating the presence

o Hb-Constant Spring. D. Hb-Barts hy rops etalis.
T ere is no Hb A present. T e majority o the hemoglo-

bin is Hb-Barts, with a small amount o Hb-Portlan .

(Reprinte rom McClatchey KD. Clinical Laboratory

Medicine, 2n e , Phila elphia, PA: Lippincott Williams
& Wilkins, 2002, with permission.)

Citrate Agar Electrophoresis Tis test involves aci enaturation o hemoglobin. Fetal

T is process takes place at an aci pH. In this metho , hemoglo- hemoglobin resists enaturation, whereas a ult hemoglobin

bins are separate on the basis o a complex interaction between oes not. Occasionally, interme iate (partially enature )

hemoglobin, agar, an citrate bu er ions. Citrate agar separates cells may be seen that are almost surely etal hemoglobin–

hemoglobin ractions that migrate together on cellulose acetate. containing cells. Elevate levels o hemoglobin F can be seen

Tese ractions are hemoglobins S, D, G, C, E, an O. All hemo- in beta-thalassemia, a orm o anemia, an paroxysmal noc-

globin specimens that show an abnormal electrophoretic pat- turnal hemoglobinuira (PNH).

tern in alkaline me ia shoul un ergo electrophoresis on aci Chromatography

citrate agar. T e combine in ormation allows or a complete Quantitation o hemoglobin A can be accomplishe by cation

i entif cation o many variant hemoglobins. 1
exchange minicolumn chromatography. However, the results

Denaturation Procedures o this technique can be a ecte by several types o hemoglo-

A proce ure commonly use to etermine the amount bin in a ition to hemoglobin A . Cellulose acetate an citrate
1
o etal bloo that has mixe with maternal bloo ollow- agar electrophoresis shoul be use in conjunction with cat-

ing elivery is the Kleihauer-Betke (Fig. 32.8) proce ure. ion exchange chromatography to eliminate the possibility o
inter erence by hemoglobin variants. Other assay metho s or

glycosylate hemoglobin inclu e high-pressure liqui chroma-
tography (HPLC) an colorimetric metho s.

Analysis of Hemoglobin

■ Several techniques have been evelope to stu y hemo-
globin inclu ing hemoglobin electrophoresis, solubility

testing, an enaturation o hemoglobin through the use

o aci or alkaline solutions.

■ Alkaline electrophoresis is a metho o separating hemo-
globin ractions base on the principle that hemoglobin

molecules in an alkaline solution have a net negative

charge an move towar the ano e in an electrophoretic

system.

FIGURE 32.8 Kleihauer-Betke stain. Kleihauer-Betke aci elu- ■ Various me ia an i erent bu ers may be use or elec-
tion or etal hemoglobin (Hb). Re bloo cells containing HbF trophoresis. For example, cellulose acetate at alkaline pH

are eeply staine re ; re cells containing HbA appear as pale is rapi an repro ucible.

pink ghosts. (Reprinte rom Greer JP (e ). Wintrobe’s Clinical ■ A proce ure use to etermine the amount o etal bloo
Hematology, 11th e , Phila elphia, PA: Lippincott Williams & that has mixe with maternal bloo ollowing elivery is

Wilkins, 2004, with permission.) the Kleihauer-Betke proce ure.