Manual of basic techniques for a health laboratory - libdoc.who.int

292 Manual of basic techniques for a health laboratoryCorrection for nucleated erythrocytesNucleated erythrocytes or normoblasts (see Fig. 9.90) are early stages of erythrocytes.They are not normally present in the blood, but they may be present in theblood in certain diseases such as sickle cell anaemia or other haemolytic anaemias.Normoblasts are not haemolysed in the diluting fluid and are therefore countedwith the leukocytes. When normoblasts are present in large numbers and theleukocytes are counted using a fully or semi-automated cell counter, the leukocytenumber concentration must be corrected as follows.Examine a thin Romanowsky-stained blood film (see section 9.10) and count thenumber of normoblasts seen for every 100 leukocytes.Calculation:The number concentration of normoblasts (per litre) is:number of normoblasts counted¥ leukocyte number concentration100 + number of normoblasts countedExample:If 50 normoblasts are counted and the leukocyte number concentration is 16 ¥ 10 9 /l,the number concentration of normoblasts is:50¥ 16 = 5. 3 ¥ 109 l100 + 50and the corrected leukocyte number concentration is:(16 - 5.3) ¥ 10 9 /l = 10.7 ¥ 10 9 /l.9.7 Measurement of the erythrocyte sedimentation rate9.7.1 PrincipleBlood collected into an anticoagulant is placed in a long graduated tube held in avertical position. The erythrocytes settle to the bottom, leaving a layer of plasmaabove.The height of the column of plasma after 1 hour indicates the sedimentation rate ofthe erythrocytes (erythrocyte sedimentation rate (ESR)).9.7.2 Materials and reagents (Fig. 9.45)●Westergren ESR tube: internal diameter 2.5mm; graduated from 0 to 200mm(often marked 1 to 20: 1 corresponds to 10mm, 2 to 20mm, etc.)●●●●●Westergren standTest-tubesGraduated syringe, 5mlGraduated pipette, 5mlTimer●Anticoagulant: trisodium citrate, 3.2% solution (reagent no. 60) (keep in refrigerator)or EDTA dipotassium salt, 10% solution (reagent no. 22).9.7.3 Method1. Pipette 0.4ml of trisodium citrate solution into a test-tube or bottle.2. Collect a venous blood specimen (see section 9.2). Apply the tourniquet as looselyas possible; puncture the vein at once and release the tourniquet.Collect 2ml of blood into a syringe.