Counting of rbc and wbc

TEC, TLC and DLC

1. Counting of RBC & WBC Submitted by Assad Al Imran 19-VMK-57

2. Total Erythrocyte Count Total erythrocyte count is a method for determining the functional state of the erythron . ( Erythron is a term for the mass of circulating erythrocytes plus the erythropoietic tissue of bone marrow ) However, this count reflects only the total number of red blood cells (RBC) in the circulating blood and does not indicate the oxygen-carrying capacity or amount of hemoglobin ( Hb ).

3. TECHNIQUES OF ERYTHROCYTE EXAMINATION Blood Collection Methods: 1. Haemocytometer Method 2. Electronic Methods 3. Using Haemoglobin Determinations Acid Haematin Method Direct Matching Method Oxyhaemoglobin Method Cyanmethaemoglobin Method

4. Collection of blood The usual procedure in large animals is to collect blood directly from the jugular vein into a test tube or vial containing an adequate amount of anticoagulant for the amount of blood desired. 5 ml of blood is sufficient for most hematologic examinations. An alternate technique in the cow is to utilize the caudal vein or artery as a source of peripheral blood , and in this instance a clean, dry syringe or Vacutainer is utilized. Blood sampling from the dog is best accomplished by use of a Vacutainer or, alternatively, a dry syringe into which has been placed one drop of anticoagulant .

5. Care must be taken in obtaining blood, as improper collection procedures may cause cellular distortion. Obtaining venous blood from a cat using a Vacutainer or dry syringe may prove difficult because of the size of the vein and the necessity for restraint. Adequate samples can be collected from the jugular vein. There are certain advantages in using the jugular vein: I t is relatively easy to locate, T here is no excessive resistance to restraint, V ein is large and easy to penetrate, and adequate quantities of blood are readily obtained, even from small animals.

6. Blood may be obtained from the ear vein of dogs and cats by first cleaning the surface of the ear followed by placing a thin layer of petroleum over the vein to be punctured. The vein should be punctured by a sharp, pointed scalpel blade or sterile disposable needle through the petroleum and into the vein. The blood forms a drop on the petroleum and is not contaminated with hair or dander from the ear. An adequate flow of blood usually results, and the sample can be either collected in diluting pipettes or utilized for other techniques.

7. An alternate technique is to clip a toenail into the vascular area. The quantity of blood collected from the ear or toenail is usually inadequate for use in automated cell counters but may be adequate for haemocytometer counts. Blood samples from swine are removed from either an ear vein or the anterior vena cava. In collecting blood, care should be taken to ensure that the needle is of sufficient diameter.

8. A needle of too small gauge may cause disruption of erythrocytes and damage leukocytes. Blood should flow smoothly with a minimum of vacuum, and if a syringe is used, one should avoid pumping the syringe barrel. If it is necessary to transfer the blood from a syringe into a test tube, the needle should be removed, as forcing blood through the needle may damage cells

9. Anticoagulants:

10. GREEN : Sodium  heparin  or lithium heparin used for plasma determinations in clinical chemistry (e.g. urea and electrolyte determination). Sodium heparin collection tubes are the classically preferred tube for peripheral blood or bone marrow for cytogenetic studies. Lithium heparin is considered suboptimal for cytogenetics . Light green or green / gray " tiger ": for plasma determinations . Purple or lavender : K 2  EDTA . This is a strong anticoagulant and these tubes are usually used for complete blood counts (CBC). Lavender top tubes are generally used when whole blood is needed for analysis. Can also be used for some blood bank procedures such as blood type and screen. EDTA tubes are preferred by most molecular genetics laboratories for molecular genetic studies (DNA or RNA ).

11. Grey : Sodium fluoride  and  oxalate . Fluoride prevents enzymes in the blood from working, by preventing glycolysis so a substrate such as glucose will not be gradually used up during storage. Oxalate is an anticoagulant. Light blue : Sodium citrate . Citrate is a reversible anticoagulant, and these tubes are used for coagulation assays. Dark blue : EDTA . These tubes are used for trace metal analysis. Black: used for Erythrocyte Sedimentation Rate (ESR).

12. 1. Haemocytometer method Equipment necessary for performing a total erythrocyte count includes: 1. Neubauer Counting chamber. 2. Special coverglass for the counting chamber. 3. RBC pipette. 4. Diluting fluid ( Haeyem’s solution or NSS 0.85%). 5. Microscope and lamp. 6. A hand tally for enumeration of cells.

13. For total leukocyte count, the WBC in the area (W) are counted. The total erythrocyte count is completed by counting all cells in the squares labeled R Fig: A Neubauer Counting Chamber focussed on a microscope (10X)

14. Fig: A Neubauer Counting Chamber Fig: An RBC pipette Fig: Counting Procedure (zig-zag) method (40X)

15. Procedure: Blood should be carefully drawn to the 0.5 mark of the RBC pipette The diluting fluid should be drawn to the 101 mark to dilute the blood The blood and diluting fluid are mixed by shaking the pipette vigorously in a horizontal position for 2 – 3 minute to ensure complete haemolysis of WBC 2 – 4 drops of mixed fluid are discarded at the end of pipette.

16. The tip of the pipette is touched to the side of the haemocytometer chamber and drop of a fluid wall run under the coverglass Wait for about 2 – 3 minutes as erythrocytes require settling time to assume a single level Total number of cells in 5 squares in the centre of counting chamber is determined under the high power of the microscope (40X)

17. Counting is Zigzag manner in all 5 inner squares Blue cells are counted Gray cells are not counted

18. Counting : TEC/ μL or mm 3 = No. of cells in 5 squares (80 small squares) x dilution no. x depth x area Dilution no. = 0.5 : 100 = 200 TEC = no. of cells counted x 1/10 depth x 1/5 mm 2 area counted x 200 dilution = No. of cells counted x 5 x 10 x 200/ μL or mm 3 = No. of cells counted x 10000/ μL or mm 3

19. 2. Electronic method Erythrocytes may be counted electronically. The high cost of some units may preclude their use in most veterinary hospital laboratories. However, the accuracy of erythrocyte counts completed by this technique is much greater than can be attained by the hemocytometer method. In some medical laboratories electronic counters are calibrated for human blood and can be used for animal bloods only if adjusted to account for the different sizes of animal erythrocytes.

20. Acid haematin method These method depend upon the conversion of hemoglobin to acid haematin, which is usually accomplished by the use of dilute hydrochloric acid. The resulting brownish-yellow mixture is matched with a standard in a colorimeter or comparator.

21. Procedure: In these techniques 0.1 N HCl is added to whole blood , and the mixture allowed to stand until acid haematin has developed. In this method, the color of the blood and acid mixture is compared with a standard, and the reading is made either in percentage of normal or in grams per deciliter of blood. Since each technique may have a different hemoglobin concentration as 100 per cent, hemoglobin should always be reported in grams per deciliter.

22. Sources of error in the acid haematin methods are numerous and include the following: N on-haemoglobin substances such as proteins and lipids normally present in plasma and cell stroma may influence the color of the diluted blood. (2) It is difficult to match the sample accurately with a brown glass standard, although this is probably the simplest glass standard available. (3) The variation in ability of individual operators in matching colors is a common source of error.

23. (4) Each match must be made after the same interval for which the instrument was standardized; failure to observe this rule introduces another error. (5) Some haemoglobin present in blood is in an inactive form as methemoglobin, sulfhaemoglobin , or carboxyhemoglobin; in acid solutions these are not converted into haematin and, consequently, are not included in values obtained by this technique. For these reasons the acid haematin methods for hemoglobin estimation are rarely used.

24. Direct Matching Method Two principal types of direct matching methods are used, Tallqvist hemoglobin scale Dare hemoglobinometer

25. Tallqvist method A drop of blood is placed on a piece of white absorbent paper, and the paper with its drop of blood is inserted in the central perforation of a serial red color chart. The intensity of red is matched with the most suitable color on the paper scale, and hemoglobin is read as the value that this scale represents. This is a rapid, simple, and inexpensive technique and should only be used as a screening procedure under field conditions, when a more accurate method is not practical.

26. Dare haemoglobinometer A drop of blood is placed in a capillary chamber between small glass plates and matched with a permanent red glass standard. This has some advantage over the Tallqvist technique, as the thickness of the drop of blood can be more carefully controlled, but comparison of red colors remains a difficult problem. The error for both the Tallqvist hemoglobin scale and Dare hemoglobinometer is estimated to be between ±10 and 40 per cent. Both techniques must be considered screening tests only.

27. Oxyhaemoglobin method: One of the simplest methods of determining oxyhemoglobin employs the Spencer haemoglobinometer . This instrument uses a green filter to measure oxyhemoglobin by light absorption. Fig: A Spencer haemoglobinometer

28. Procedure: A drop of blood is placed on a glass plate, and cells are laked by a hemolytic agent. The usual hemolytic agent is saponin , which is dried on the end of an applicator stick. The thickness of the drop of blood is controlled by placing a second glass plate over the one containing laked blood and pressing the two together. This glass chamber is inserted into the haemoglobinometer, and the green color is matched with that of a standard .

29. Green colors are somewhat easier to match than other shades, consequently this technique is relatively more accurate than those involving other colors. Green also has the advantage that the maximum absorption of hemoglobin under visual light occurs in the green band of the spectrum.

30. Cyanmethaemoglobin method: This is probably the most accurate and widely used technique for determining hemoglobin concentration Fig: A spectrophometer Requirements : Spectrophotometer Sahli’s pipette 20 μL Pipette 5 mL

31. Procedure: Take 5 ml of Drabkins solution and to it add 20 μL of blood Stopper the tube, mix by inverting several times Allow to stand for 5 minutes Transfer the sample to cuvette Read the absorbance in the spectrophotometer at 540 nm Also take the absorbance of the standard solution

32. A graph can be plotted when a large number of samples are processed Hb concentration on horizontal axis and absorbance on vertical axis Result :

33. Normal values of TEC in different animals:

34. Total Leukocyte Count: To enumerate the total number of leukocyte (WBC) of a given blood sample. Equipment necessary for performing a total leukocyte count includes: 1. Neubauer Counting chamber. 2. Special coverglass for the counting chamber. 3. WBC pipette. 4. Diluting fluid (1% HCl or 1% glacial acetic acid). 5. Microscope and lamp. 6. A hand tally for enumeration of cells.

35. Fig: A Neubauer Counting Chamber Fig: A WBC pipette Fig: Counting Procedure (zig-zag) method (40X)

36. Procedure: Blood should be carefully drawn to the 0.5 mark of the WBC pipette The diluting fluid should be drawn to the 11 mark to dilute the blood The blood and diluting fluid are mixed by shaking the pipette vigorously in a horizontal position for 2 – 3 minute to ensure complete haemolysis of RBC 2 – 4 drops of mixed fluid are discarded at the end of pipette.

37. The tip of the pipette is touched to the side of the haemocytometer chamber and drop of a fluid wall run under the coverglass Wait for about 2 – 3 minutes as leukocytes require settling time Total number of cells in 4 squares on the outside of counting chamber is determined under the low power of the microscope (10X)

38. Counting is Zigzag manner in all 4 outer squares Blue cells are counted Gray cells are not counted

39. Counting : TLC/ μL or mm 3 = No. of cells in 4 squares (64 small squares) x dilution no. x depth x area Dilution no. = 0.5 : 10 = 20 TLC = No. of cells counted x 10 x ¼ x 20/ μL or mm 3 = No. of cells counted x 50/ μL or mm 3

40. Differential Leukocyte Count: It represents percentage of various leucocytes in stained smear or bone marrow Requirement: Clean slides Leishman’s stain/Wright’s stain/Giemsa’s stain Blood cell couter

41. Procedure: A thin blood smear is made from whole blood A good smear should be tongue shaped without tails After proper drying the smear it is stained and air dried. The edge of the smear is examined under oil immersion.

42. Moving the slide vertically and horizontally, a total of 100 leucocyte are counted using blood cell counter. Four fields at edges, centre and tail may be counted. Different types of leucocyte are expressed in percentage The absolute values are calculated for each of the leucocyte after enumerating TLC of the sample.

43. Counting : For e.g., out of 100 leucocytes counted 56 were neutrophils. Then, 56% is the DLC value of neutrophil Absolute neutrophil count = 56/100 x 10,000/cu mm =5,600/cu mm

44. References: Veterinary Clinical Pathology 4 th Edition - Coles, Embert H https://www.slideshare.net/ParasuramanParasuraman/rbc-count-and-wbc-count?qid=b2bd725d-f9e9-41a6-ac90-ee6b8e653bb8&v=&b=& from_search=1 VLD Manual ( CVSc ) Google.com https://en.wikipedia.org/wiki/Vacutainer

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