1. Determination of Hb values M. Ss. Program CLS 541 Lab-3-

2. Determination of Hb values Haemoglobin can be measured by manual or automatic. Two method are common use: 1- haemiglobincyanid. (HicN;Cyanmethaemoglobin ) 2-Oxyhaemoglobin (Hbo 2 method ).

3. A major a advantage of the HicN method is availability of stable and reliable reference preparation. Other methods: Sahli’s acid haematin and alkalin-haematin method.

4. Cyanmethaemoglobin method Use Drabkins solution which contain potassium cyanide and potassium ferricynide at PH7-7.4. Hb oxidation to methaemoglbin by potassium ferricynide then combine to potassium cyanide to form cyanmethaemoglobin which measure at 540 nm by spectrophotometer.

5. Normal rage of Hb in g/dl Hb g/dl= (Ax64500xD. F)÷(44XDx1000x10) Men 15 ± 2 g/dl Women 13.5 ± 1.5g/dl At birth 18 ± 4g/dl

6. R.B.C, W.B.C and Platelet count The cell blood count can used manual or automatic. A variety of automated instruments for performing blood counts have been developed and are now widespread use. A variety of automated instruments for performing blood counts have been developed and are now widespread use. Manual cell counts using counting chambers: it is not recommended for routine because will be too few to ensure a precise.

7. Improved Neubauer counting chamber contain 9 1mmx1mm ruled areas each contain a volume of 0.1µl of diluted blood examine at 40x objective. For W.B.C count use the acidic acid with violet stain as the diluents but for R.B.C and platelets use the formal citrate as the diluents.

8. Normal rage for R.B.C count For men: 5 ± 0.5 x10 12 /L. For women: 4.3 ± 0.5 x10 12 /L. Normal range of W.B.C count For men and women: 7.0 ± 3.0X10 9 L. Normal range of platelets count For men and wmen: 150-400 x 10 9 L.

9. Packed Cell Volume (PCV) determination and red cell indices Two methods: 1-macro-method of wintrobe which is no longer in routine use. 2-micro-haemotocrite which the centrifuge used for capillary tubes provides a centrifugal force of c12000g and 5min.

10. PCV for men: 0.45 ± 0.05 L/L. PCV for women: 0.41 ± 0.05 L/L. PCV for infant: 0.60 ± 0.15 L/L. MCV= (PCV%X10) ÷ R.B.C. MCV:92 ± 9 FL. MCH= (Hbg/dlx10) ÷ R.B.C. MCH: 29.5 ± 2.5 Pg. MCHC= (Hb ÷ PCV) X 100. MCHC: 33% ± 1.5% 0R g/dL.

11. ESR DETERMINATION The recommended method is Westergren method. The test is performed on venous blood. It conventional to set up sedimentation rate tests at room temperature(18-25°C). The usual practice is to collect the blood dirctly into the citrate solution. The test can be carried out equally well with blood anticoagulated with EDTA within24h.

12. If the specimem is kept at 4°C,provided that 1volume of 109mmol/l(23g/l) trisodium citrate is add to 4volumes of blood immediately befor the test is performed. the blood will draw it up into the Westergren tube to 200mm mark, place the tbe exactly vertical and leave undistrubed for exactly 60min.the result expressed as ESR=X min in 1h. ESR for men 70=c30). ESR for women 70=c35).

13. Reticulocyte count Reticulocytes are immature red cells. They contine remnants of ribosomal ribonucleic acid. To stain the reticulocytes use basic dyes such as azure B,brilliant creslyle blue or new methylene blue to form a blue or purple precipitate of granules of filments (unfixed prepartion). reticulocytes can be divided into four groups according maturation (group I until groupIV).

14. The group I which contain a large clump of reticun but group IV with few granules of reticulin group. The ripening process in thought to take 2-3 days. The number of reticulocytes in peripheral blood is a fairly accurate reflection of erythropoetic activity. Normal = 0.05 - 2.5%. Infant = 2 - 5%.