Page 699 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

CHAPTER 32 ■ Laboratory Manual: Manual Procedures in Hematology 683

SPECIAL HEMATOLOGY PROCEDURES (continued)

■ Chromatography can be use to quantitate hemoglobin o solution A an 73.3 mL o solution B. Bu er (6.8 pH).

A . Assay metho s or glycosylate hemoglobin inclu e Mix 49.6 mL o solution A an 50.4 mL o solution B.
1
high-pressure liqui chromatography (HPLC) an colo- Refrigerate all solutions to store.

rimetric metho s. 7. Giemsa stock stain: Prepare by a ing 5 g o pow ere

Giemsa stain to 330 mL o reagent-gra e glycerol. Mix.
HEMOGLOBIN S SCREENING TEST: QUALITATIVE Place in a 60°C oven or 2 hours. Allow to cool. Mix. With

DIFFERENTIAL SOLUBILITY TEST constant stirring, slowly a 330 mL o methanol. rans er

Principle to a stoppere brown bottle an shake or a ew minutes.

Tis is a biphasic system consisting o an upper organic phase Label. Filter before use.

o toluene an a lower, aqueous phase containing phosphate Working Giemsa Stain: Prepare by a ing 10 mL o f ltere

bu er, saponin, an re ucing agents. Erythrocytes are lyse Giemsa stock stain to 90 mL o bu er solution. Mix.

by toluene an saponin, with the release hemoglobin being

re uce by so ium hy rosulf te. T e resulting colors o the 1. Coplin or other type o staining jar an sli e hol er

aqueous phase an the inter ace phase allow or the i eren- 2. Microscope, immersion oil, an lens paper

tiation o hemoglobin types AA, AS, an SS. Quality Control

Detection o the abnormal Hb S is iagnostic o sickle cell

isease. Hb S, i inherite in the homozygous state (SS), results A re erence sli e set shoul be maintaine to vali ate the

in sickle cell anemia. Inheritance o the heterozygous state ability o microscopists to i enti y various malarial spe-

(AS) pro uces a benign an asymptomatic con ition, except cies. Participation in a quality assurance program, such as

un er con itions o re uce oxygen levels. T e etection o the College o American Pathologists program, is impor-

this heterozygous state is important to iagnose in in ivi uals tant in maintaining expertise in this area, particularly in

who are involve in strenuous physical sports, such as long- laboratories that in requently encounter positive results.

istance runners, or in in ivi uals whose occupations have Procedure

the potential or re uce oxygen levels, such as test pilots.

Tis proce ure, in CLSI ormat, is provi e on this book’s 1. Following bloo smear preparation, allow the smears to

companion Web site at http://thepoint.lww.com/ urgeon6e. air- ry or approximately 30 minutes. Fix the thin smears
in methanol or a ew secon s. Do not f x the thick smears.

MALARIAL SMEARS 2. Place the smears in a Coplin jar containing Giemsa stain-

Principle ing solution or 30 minutes. Rinse the smears in running
tap water an allow to air- ry.
Tick an thin bloo smears are prepare , staine , an exam- 3. Examine the smears using the oil immersion objective.

ine microscopically or the presence o one o our malaria Te erythrocytes on the thick, unf xe smears will be

types. Detection an correct i entif cation o the species o estroye , making examination easier. T e thick smear is

malaria (Plasmodium malariae, P. vivax, P. falciparum, or use as a screening test to establish the presence o the

P. ovale) are important to ensure proper treatment.
parasite. T e thin smear, which allows or care ul exami-

Specimen nation o cellular morphology, permits i entif cation o

Smears o capillary or ED A-anticoagulate bloo are pre- the species o malaria.

pare as escribe in the section on specimen preparation Reporting Results

in Chapter 2.
Te iagnosis o malaria is base on the emonstration o

Reagents, Supplies, and Equipment the Plasmodium species in the bloo . Re er to Figure 32.9

1. American Chemical Society (ACS)-gra e methanol or illustrations o the typical appearance o various species.

2. ACS-gra e glycerol A brie morphological escription is given in able 32.2. For

3. Bu er (pH 6.4 or 6.8) a complete iscussion o each o the Plasmodium species, as

4. Solution A: Prepare by placing 9.47 g o anhy rous secon ary well as other actors relate to malaria, see Chapter 7.

so ium phosphate (Na HPO ) or 11.87 g o hy rate so ium
2
4
phosphate (Na HPO ·2H O) into a 1-L volumetric ask. Procedure Notes
2
4
2
Dilute to the calibration mark with eionize water. Mix. Sources of Error
5. Solution B: Weigh an trans er 9.080 g o primary potas- Malarial parasites can be con use with platelets. It is

sium phosphate (KH PO ) into a 1-L volumetric ask. important to istinguish between malarial parasites in the
2
4
Dilute to the calibration mark with eionize water. Mix. erythrocyte an platelets that are superimpose on the eryth-

6. Solutions A an B can be mixe in various proportions rocyte. Malarial parasites are never seen in the spaces between

to achieve a i erent pH. Bu er (6.4 pH). Mix 26.7 mL erythrocytes.

(continued)