Page 697 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

CHAPTER 32 ■ Laboratory Manual: Manual Procedures in Hematology 681

SPECIAL HEMATOLOGY PROCEDURES (continued)

nucleoti es, when activate with long-wave (340 to 370 nm)
Cell Type Mean %
ultraviolet (UV) light, is use or visual examination o G6PD

Metarubricytes 6.5 activity. T e observe rate o the appearance o bright uo-
rescence is proportional to the bloo G6PD activity. G6PD
Neutrophil series 56.0
ef ciency is one o the most prevalent here itary erythrocyte
Blasts 1.0 enzyme ef ciencies. A ef ciency o this enzyme can pro uce

Promyelocytes 3.4 rug- or stress-in uce hemolytic anemia in a icte persons.

Myelocytes 11.9 T is proce ure, in CLSI ormat, is provi e on this book’s
companion Web site at http://thepoint.lww.com/ urgeon6e.
Metamyelocytes 18.0

Bands 11.0 HEMOGLOBIN ELECTROPHORESIS: CELLULOSE

ACETATE METHOD
PMNs 10.7
Principle
Eosinophil series 3.2
Electrophoresis may be ef ne as the movement o charge
Basophils <0.1 particles on various me ia un er the in uence o an electric

Lymphocytes 15.8 current. Particles move at i erent spee s because o their

Monocytes 1.8 weight an electric charge.
In hemoglobin electrophoresis, a hemolysate prepare rom
Megakaryocytes <0.1
intact erythrocytes is place on a me ium such as cellulose
Reticulum cells 0.3
acetate. T e strips o cellulose acetate are place in an alka-

Plasma cells 1.8 line bu er (pH 8.0 to 8.6), an electrical charge is applie . T e

M/E ratio 2.5:1 strips are staine to see the hemoglobin ractions. A compari-
son o the unknown hemolysate with hemolysates rom known

hemoglobin types is ma e. Hemoglobin electrophoresis by cel-

BIBLIOGRAPHY lulose acetate is use ul in i enti ying an quanti ying hemoglo-

bin variants an abnormal quantities o hemoglobin ractions.

Provi e on this book’s companion Web site at http://thepoint. Tis proce ure, in CLSI ormat, is provi e on this book’s

lww.com/ urgeon6e. companion Web site at http://thepoint.lww.com/ urgeon6e.

DONATH-LANDSTEINER SCREENING TEST Analysis of Hemoglobin

Principle Since the f rst hemoglobinopathy was escribe in 1910, sev-

T e Donath-Lan steiner antibo y test is use to emonstrate eral techniques have been evelope to stu y hemoglobin.

the presence o this extremely potent hemolysin. T is antibo y Tey inclu e hemoglobin electrophoresis with various me ia,

requires col incubation to exhibit hemolysis in the patient’s such as paper, cellulose acetate, or agar; solubility testing; an

serum. A positive result is iagnostic o paroxysmal col hemoglo- enaturation o hemoglobin through the use o aci or alka-

binuria (PCH), the rarest orm o autoimmune hemolytic anemia. line solutions (see Chapter 17). A metho or the preliminary

T is proce ure, in CLSI ormat, is provi e on this book’s i entif cation o abnormal hemoglobin is electrophoresis.

companion Web site at http://thepoint.lww.com/ urgeon6e.
Alkaline Electrophoresis

GLUCOSE-6-PHOSPHATE DEHYDROGENASE T is metho o separating hemoglobin ractions is base on

ACTIVITY IN ERYTHROCYTES: VISUAL the principle that hemoglobin molecules in an alkaline solu-

FLUORESCENT SCREENING TEST tion have a net negative charge an move towar the ano e in

Principle an electrophoretic system. T is screening proce ure can sep-

Te enzyme glucose-6-phosphate ehy rogenase (G6PD) arate hemoglobins A, F, S, an C an other variant hemoglo-

catalyzes the ollowing reaction: bins (Fig. 32.7). T ose with a greater electrophoretic mobility
than hemoglobin A at pH 8.6 are classif e as the ast hemo-

6
Glucose- -phosphate + NADP GPD → globins. Examples o ast hemoglobins are Bart hemoglobin
6
( not luorescent ) an the two astest variants, hemoglobin H an hemoglobin
+
6 -phosphoggluconate NADPH I. Hemoglobin C is the slowest common hemoglobin.
( not luorescent )
Various me ia, such as paper, cellulose acetate, or starch blocks,

Te reaction mixture containing glucose-6-phosphate, an i erent bu ers may be use or electrophoresis. T ese alter-

nicotinami e a enine inucleoti e phosphate (NADP), native metho s vary in their e ciency o separation. For example,

an bloo is incubate , an at time intervals, rops o the cellulose acetate at alkaline pH is rapi an repro ucible. T is

mixture are applie to f lter paper. T e uorescence o re uce technique separates the hemoglobin ractions S, F, A, C, an A .
2

(continued)