Counting Cells Using Manual Methods PDF

Counting cells using manual methods 2nd Practical pathological analysis :‫اﻟﻄﺮق اﻟﻴﺪوﻳﺔ ﻟﺤﺴﺎب اﻟﺨﻼﻳﺎ‬ Manual methods of cell counting: EDTA should be used EDTA ‫ﻳﺠﺐ اﺳﺘﺨﺪام‬...

Counting cells using manual methods 2nd Practical pathological analysis :‫اﻟﻄﺮق اﻟﻴﺪوﻳﺔ ﻟﺤﺴﺎب اﻟﺨﻼﻳﺎ‬ Manual methods of cell counting: EDTA should be used EDTA ‫ﻳﺠﺐ اﺳﺘﺨﺪام‬ :‫اﻟﻄﺮق اﻟﻴﺪوﻳﺔ اﻟﻤﺴﺘﺨﺪﻣﺔ ﻓﻲ‬ Manual methods used for: 1. check on the validity of electronic methods for calibration purposes 2. As a check on the validity of electronic counts in patients with leukopenia or thrombocytopenia 3. For blood specimens with platelet counting interference (i.e., very microcytic RBCs) It is also commonly used as a method for counting cells in cerebrospinal fluid (CSF) and many body fluids such as seminal fluid. ‫ اﻟﺘﺤﻘﻖ ﻣﻦ ﺻﺤﺔ اﻟﻄﺮق اﻹﻟﻜﺘﺮوﻧﻴﺔ ﻷﻏﺮاض اﻟﻤﻌﺎﻳﺮة‬.1 ‫ ﻟﻠﺘﺤﻘﻖ ﻣﻦ ﺻﺤﺔ اﻟﻌﺪ اﻹﻟﻜﺘﺮوﻧﻲ ﻟﺪى اﻟﻤﺮﺿﻰ اﻟﺬﻳﻦ ﻳﻌﺎﻧﻮن ﻣﻦ ﻧﻘﺺ اﻟﻜﺮﻳﺎت اﻟﺒﻴﺾ أو ﻧﻘﺺ اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ‬.2 (‫ ﻟﻌﻴﻨﺎت اﻟﺪم اﻟﺘﻲ ﺗﻌﺎﻧﻲ ﻣﻦ ﺗﺪاﺧﻞ ﻓﻲ ﺗﻌﺪاد اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ )أي ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء ﺻﻐﻴﺮة اﻟﺤﺠﻢ ﺟﺪً ا‬.3  :‫اﻟﻌﻴﻮب اﻟﺮﺋﻴﺴﻴﺔ ﻟﺤﺴﺎب اﻟﺨﻼﻳﺎ اﻟﻴﺪوي ﻫﻲ‬ The main drawbacks to manual cell counting are: It requires human interpretation of what are cells and assessment of live/dead cells, causing high error rates between operators. Slow: Counting and recording large numbers of cells is a laborious, time-consuming process Hemocytometers must be thoroughly cleaned between samples. Volumetric errors: Pipetting errors and poor siting of the cover slip can lead to measurement inaccuracies..‫ ﻣﻤﺎ ﻳﺘﺴﺒﺐ ﻓﻲ ارﺗﻔﺎع ﻣﻌﺪﻻت اﻟﺨﻄﺄ ﺑﻴﻦ اﻟﻤﺸﻐﻠﻴﻦ‬،‫اﻟﻤﻴﺘﺔ‬/‫ﺗﻔﺴﻴﺮا ﺑﺸﺮﻳً ﺎ ﻟﻤﺎﻫﻴﺔ اﻟﺨﻼﻳﺎ وﺗﻘﻴﻴﻢ اﻟﺨﻼﻳﺎ اﻟﺤﻴﺔ‬ ً ‫ ﻳﺘﻄﻠﺐ‬ ً ‫ ﻳﻌﺪ ﻋﺪ وﺗﺴﺠﻴﻞ أﻋﺪاد ﻛﺒﻴﺮة ﻣﻦ اﻟﺨﻼﻳﺎ ﻋﻤﻠﻴﺔ ﺷﺎﻗﺔ وﺗﺴﺘﻐﺮق وﻗﺘً ﺎ ﻃﻮﻳ‬:‫ ﺑﻄﻲء‬ ‫ﻼ‬.‫ ﻳﺠﺐ ﺗﻨﻈﻴﻒ ﻋﺪادات اﻟﺪم ﺟﻴﺪً ا ﺑﻴﻦ اﻟﻌﻴﻨﺎت‬.‫ ﻳﻤﻜﻦ أن ﺗﺆدي أﺧﻄﺎء اﻟﻤﺎﺻﺔ ووﺿﻊ اﻟﻐﻄﺎء ﻏﻴﺮ اﻟﺼﺤﻴﺢ إﻟﻰ ﻋﺪم دﻗﺔ اﻟﻘﻴﺎس‬:‫ أﺧﻄﺎء اﻟﺤﺠﻢ‬.‫ ﺷﺮﻳﺤﺔ ﺧﺎﺻﺔ ﺑﻤﺴﺎﺣﺔ ﻣﺤﺴﻮﺑﺔ وﻋﻤﻖ ﻣﻊ درﺟﺔ اﻟﻤﺮﺑﻌﺎت‬ special slide with calculated area and Counting chamber: depth with grade of squares. :‫ﻏﺮﻓﺔ اﻟﻌﺪ‬ Diluting solution: according to cell type..‫ ﺣﺴﺐ ﻧﻮع اﻟﺨﻠﻴﺔ‬ :‫ﻣﺤﻠﻮل ﻣﺨﻔﻒ‬ For viability test: Manual cell counting dye such as trypan blue used. can be performed Cells are mixed with the dye and incubated with or without stains. Trypan blue is used to count dead cells; ‫ﻳﻤﻜﻦ إﺟﺮاء ﻋﺪ اﻟﺨﻼﻳﺎ ﻳﺪوﻳً ﺎ‬.‫ﻣﻊ أو ﺑﺪون ﺑﻘﻊ‬ :‫ ﻻﺧﺘﺒﺎر اﻟﻘﺪرة ﻋﻠﻰ اﻟﺒﻘﺎء‬.‫ ﻳﺘﻢ اﺳﺘﺨﺪام ﺻﺒﻐﺔ ﻣﺜﻞ اﻷزرق اﻟﺘﺮﻳﺒﺎن‬.‫ ﻳﺘﻢ ﺧﻠﻂ اﻟﺨﻼﻳﺎ ﻣﻊ اﻟﺼﺒﻐﺔ وﺣﻀﻨﻬﺎ‬ ‫ ﻳﺘﻢ اﺳﺘﺨﺪام اﻷزرق اﻟﺘﺮﻳﺒﺎن ﻟﺤﺴﺎب اﻟﺨﻼﻳﺎ اﻟﻤﻴﺘﺔ؛‬  ‫ﻣﺘﻄﻠﺒﺎت اﻻﺳﺘﻘﺮار‬ Stability Requirements Temperature Period Causes for Rejection 1. Hemolysis; Room temperature 1 day 2. clotted specimen; Refrigerated 3 days 3. tube not filled with minimum volume; Frozen Unstable 4. improper labeling; Freeze/thaw cycles Unstable 5. specimen received in any anticoagulant other than EDTA; 6. specimen received with plasma removed; ‫ اﻧﺤﻼل اﻟﺪم؛‬.1 7. specimen diluted or contaminated with IV ‫ ﻋﻴﻨﺔ ﻣﺘﺠﻠﻄﺔ؛‬.2 fluid; ‫ أﻧﺒﻮب ﻏﻴﺮ ﻣﻤﻠﻮء ﺑﺎﻟﺤﺪ اﻷدﻧﻰ ﻣﻦ اﻟﺤﺠﻢ؛‬.3 8. specimen received in whole blood ‫ ﺗﺴﻤﻴﺔ ﻏﻴﺮ ﻣﻨﺎﺳﺒﺔ؛‬.4 transport tube ‫ ﻋﻴﻨﺔ ﺗﻢ اﺳﺘﻼﻣﻬﺎ ﺑﺄي ﻣﻀﺎد ﻟﻠﺘﺨﺜﺮ ﻏﻴﺮ‬.5 ‫؛‬EDTA ‫ ﻋﻴﻨﺔ ﺗﻢ اﺳﺘﻼﻣﻬﺎ ﺑﻌﺪ إزاﻟﺔ اﻟﺒﻼزﻣﺎ ﻣﻨﻬﺎ؛‬.6 ‫ ﻋﻴﻨﺔ ﺗﻢ ﺗﺨﻔﻴﻔﻬﺎ أو ﺗﻠﻮﺛﻬﺎ ﺑﺴﺎﺋﻞ ورﻳﺪي؛‬.7 ‫ ﻋﻴﻨﺔ ﺗﻢ اﺳﺘﻼﻣﻬﺎ ﻓﻲ أﻧﺒﻮب ﻧﻘﻞ اﻟﺪم اﻟﻜﺎﻣﻞ‬.8 :‫ﻋﺪد ﻛﺮﻳﺎت اﻟﺪم اﻟﺒﻴﻀﺎء‬ Leukocyte Counts: Diluting Fluid: lyses the erythrocytes Well-mixed blood is diluted 1 : 20 in diluting fluid: 380 µL of Türck solution and 20 µL of blood. Mixed and left for about 5 minutes. chamber is loaded with enough fluid to fill it after putting cover slide. Left for 1-2 minutes to settle. Counted in the four corner large squares. ‫ ﻳﺤﻠﻞ ﻛﺮﻳﺎت اﻟﺪم اﻟﺤﻤﺮاء‬:‫ ﺳﺎﺋﻞ اﻟﺘﺨﻔﻴﻒ‬ :‫ ﻓﻲ ﺳﺎﺋﻞ اﻟﺘﺨﻔﻴﻒ‬1:20 ‫ ﻳﺘﻢ ﺗﺨﻔﻴﻒ اﻟﺪم اﻟﻤﺨﻠﻮط ﺟﻴﺪً ا ﺑﻨﺴﺒﺔ‬.‫ ﻣﻴﻜﺮوﻟﺘﺮ ﻣﻦ اﻟﺪم‬20‫ ﻣﻴﻜﺮوﻟﺘﺮ ﻣﻦ ﻣﺤﻠﻮل ﺗﻮرك و‬380.‫ دﻗﺎﺋﻖ ﺗﻘﺮﻳ ًﺒﺎ‬5 ‫ ﻳﺘﻢ اﻟﺨﻠﻂ وﻳﺘﺮك ﻟﻤﺪة‬.‫ ﻳﺘﻢ ﺗﺤﻤﻴﻞ اﻟﺤﺠﺮة ﺑﻜﻤﻴﺔ ﻛﺎﻓﻴﺔ ﻣﻦ اﻟﺴﺎﺋﻞ ﻟﻤﻠﺌﻬﺎ ﺑﻌﺪ وﺿﻊ ﺷﺮﻳﺤﺔ اﻟﻐﻄﺎء‬.‫ دﻗﻴﻘﺔ ﺣﺘﻰ ﻳﺴﺘﻘﺮ‬2-1 ‫ ﻳﺘﺮك ﻟﻤﺪة‬.‫ ﻳﺘﻢ اﻟﻌﺪ ﻓﻲ اﻟﻤﺮﺑﻌﺎت اﻟﻜﺒﻴﺮة ذات اﻟﺰواﻳﺎ اﻷرﺑﻊ‬  D.F × 10 × (‫ ﻋﺪد اﻟﻤﺮﺑﻌﺎت‬/ ‫( = )ﻋﺪد اﻟﺨﻼﻳﺎ‬3‫ ﻣﻢ‬/ ‫ ﻋﺪد اﻟﻜﺮﻳﺎت اﻟﺒﻴﻀﺎء )ﺧﻼﻳﺎ‬.10 : 1 ‫ ﻳﺘﻢ ﺗﺨﻔﻴﻒ اﻟﺪم ﺑﻨﺴﺒﺔ‬،2500 ‫ إذا ﻛﺎن اﻟﻌﺪد اﻹﺟﻤﺎﻟﻲ أﻗﻞ ﻣﻦ‬.200 : 1 ‫ أو ﺣﺘﻰ‬100 : 1 ‫ ﻗﺪ ﻳﻜﻮن اﻟﺘﺨﻔﻴﻒ‬،‫ ﻓﻲ ﺣﺎﻟﺔ زﻳﺎدة ﻋﺪد اﻟﻜﺮﻳﺎت اﻟﺒﻴﻀﺎء‬ ‫ ﻣﺎ ﻫﻲ اﻷﺳﺒﺎب اﻟﻤﺘﻮﻗﻌﺔ ﻟﻸﺧﻄﺎء ؟؟؟؟‬ Leukocyte count ( cells / mm3) = (No. Neubauer counting chambers cells / No. of squares) × D.F × 10 if a total count below 2500, the blood is diluted 1 : 10. In leukocytosis, the dilution may be 1 : 100 or even 1 : 200. What are expected causes of errors???? :‫اﻻﺧﺘﻼﻓﺎت ﺗﺴﺒﺐ‬ Variation causes: 1. Nucleated Red Blood Cells (NRBCs) correction: Could not distinguished in counting method Distinguished only in stained smear correction formula: True leukocyte count = (Total count × 100) / (100 + No. of NRBCs) No. of NRBCs counted during the enumeration of 100 leukocytes in the differential count. ‫ ﺗﺼﺤﻴﺢ ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء‬.1 ‫ ﻻ ﻳﻤﻜﻦ‬:(NRBCs) ‫اﻟﻨﻮوﻳﺔ‬ Example ‫اﻟﺘﻤﻴﻴﺰ ﺑﻴﻨﻬﺎ ﺑﻄﺮﻳﻘﺔ اﻟﻌﺪ ﻳﺘﻢ‬ The blood smear shows 25 NRBCs per 100 leukocytes. The total ‫ﺗﻤﻴﻴﺰﻫﺎ ﻓﻘﻂ ﻓﻲ اﻟﺼﺒﻐﺔ اﻟﻤﻠﻄﺨﺔ‬ nucleated cell count is 10,000. ‫ ﺗﻌﺪاد اﻟﻜﺮﻳﺎت‬:‫ﺻﻴﻐﺔ اﻟﺘﺼﺤﻴﺢ‬ True leukocyte count = 10,000 × 100/125 = 8000/μL (8.0 × 109/L) ‫اﻟﺒﻴﻀﺎء اﻟﺤﻘﻴﻘﻲ = )اﻟﻌﺪد اﻹﺟﻤﺎﻟﻲ‬ ‫ ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم‬+ 100) / (100 × ‫ﻣﺜﺎل‬ ‫اﻟﺤﻤﺮاء اﻟﻨﻮوﻳﺔ( ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم‬ ‫ ﺧﻠﻴﺔ‬100 ‫ ﺧﻠﻴﺔ دﻣﻮﻳﺔ ﻏﻴﺮ ﻧﻮوﻳﺔ ﻟﻜﻞ‬25 ‫ ُﺗﻈﻬﺮ ﺻﺒﻐﺔ اﻟﺪم‬ ‫اﻟﺤﻤﺮاء اﻟﻨﻮوﻳﺔ اﻟﺘﻲ ﺗﻢ إﺣﺼﺎؤﻫﺎ‬.10000 ‫ وﻳﺒﻠﻎ إﺟﻤﺎﻟﻲ ﻋﺪد اﻟﺨﻼﻳﺎ اﻟﻨﻮوﻳﺔ‬.‫دم ﺑﻴﻀﺎء‬ ‫ ﺧﻠﻴﺔ ﺑﻴﻀﺎء ﻓﻲ‬100 ‫أﺛﻨﺎء ﺗﻌﺪاد‬ = 100/125 × 10000 = ‫ ﻋﺪد اﻟﺨﻼﻳﺎ اﻟﺒﻴﻀﺎء اﻟﺤﻘﻴﻘﻲ‬.‫اﻟﻌﺪ اﻟﺘﻔﺎﺿﻠﻲ‬ (‫ﻟﺘﺮ‬/109 × 8.0) ‫ﻣﻴﻜﺮوﻟﺘﺮ‬/8000  ‫ﺗﻌﺪاد اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ‬ ‫ ﻳﺠﺐ أن ﻳﺘﻢ ذﻟﻚ ﺧﻼل ‪ 3‬ﺳﺎﻋﺎت ﻣﻦ ﺟﻤﻊ اﻟﻌﻴﻨﺔ ﻓﻲ درﺟﺔ ﺣﺮارة اﻟﻐﺮﻓﺔ أو ‪ 24‬ﺳﺎﻋﺔ ﻓﻲ ‪ 4‬درﺟﺎت ﻣﺌﻮﻳﺔ‪.‬‬ ‫ ﻳﺘﻢ ﺗﺨﻔﻴﻒ اﻟﺪم اﻟﻤﺨﻠﻮط ﺟﻴﺪً ا ﺑﻨﺴﺒﺔ ‪ 1:100‬ﻓﻲ ﺳﺎﺋﻞ ﻣﺨﻔﻒ‪ ،‬ﻓﻲ اﻟﺨﻼط ﻟﻤﺪة ‪ 10‬إﻟﻰ ‪ 15‬دﻗﻴﻘﺔ‪.‬‬ ‫ ﻳﺘﻢ ﻣﻞء ﺣﺠﺮة ﻋﺪاد اﻟﻜﺮﻳﺎت اﻟﺪﻣﻮﻳﺔ وﺗﺮﻛﻬﺎ ﻟﻤﺪة دﻗﺎﺋﻖ ﺣﺘﻰ ﺗﺴﺘﻘﺮ ﺗﺤﺖ اﻟﺮﻃﻮﺑﺔ‪.‬‬ ‫ ﻳﺘﻢ ﻋﺪ اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ ﻓﻲ ‪ 10‬ﻣﺮﺑﻌﺎت ﺻﻐﻴﺮة‪ ،‬ﺧﻤﺴﺔ ﻋﻠﻰ ﻛﻞ ﺟﺎﻧﺐ ﻣﻦ اﻟﺤﺠﺮة‪.‬‬ ‫‪Platelet Counts‬‬ ‫‪ Must done Within 3 hours of collection in‬‬ ‫‪RT or 24 hour in 4 ◦c.‬‬ ‫‪ Well-mixed blood is diluted 1 : 100 in‬‬ ‫‪diluting fluid, on mixer for 10 to 15‬‬ ‫‪minutes.‬‬ ‫‪ Hemocytometer chamber is filled left for‬‬ ‫‪minutes to settle under moisture.‬‬ ‫‪ Platelets are counted in 10 small squares‬‬ ‫‪five on each side of the chamber.‬‬ ‫إذا ﻛﺎن اﻟﻌﺪد اﻹﺟﻤﺎﻟﻲ أﻗﻞ ﻣﻦ ‪،100‬‬ ‫ ﻗﻢ ﺑﻌﺪّ ﺟﻤﻴﻊ اﻟﻤﺮﺑﻌﺎت اﻟـ ‪ 25‬ﻓﻲ اﻟﻤﺮﺑﻊ اﻟﻤﺮﻛﺰي‬ ‫اﻟﻜﺒﻴﺮ ﻋﻠﻰ ﺟﺎﻧﺒﻲ ﻋﺪاد اﻟﻜﺮﻳﺎت اﻟﺪﻣﻮﻳﺔ‪ ،‬إذا ﻟﺰم اﻷﻣﺮ‪.‬‬ ‫‪If the total No. is less than 100,‬‬ ‫‪ count all 25 squares in the large central square on each‬‬ ‫‪side of the hemocytometer, if necessary.‬‬ ‫إذا ﻛﺎن اﻟﻌﺪد اﻹﺟﻤﺎﻟﻲ ﻓﻲ ﺟﻤﻴﻊ ﻫﺬه اﻟﻤﺮﺑﻌﺎت اﻟﺼﻐﻴﺮة اﻟﺨﻤﺴﻴﻦ أﻗﻞ ﻣﻦ ‪50‬‬ ‫‪If the total No. in all 50 of these small squares is less than 50,‬‬ ‫‪ the count should be repeated with a 1 : 20 or a 1 : 10‬‬ ‫‪dilution of blood.‬‬ ‫ ﻳﺠﺐ ﺗﻜﺮار اﻟﻌﺪ ﺑﺎﺳﺘﺨﺪام ﻧﺴﺒﺔ ﺗﺨﻔﻴﻒ ﻣﻦ اﻟﺪم ‪ 20 : 1‬أو ‪.10 : 1‬‬ ‫‪Calculation‬‬ ‫‪ For the 25 small squares‬‬ ‫‪ Platelet count (per μL) = (Number of cells counted/‬‬ ‫‪Number of squares counted) × Dilution × 250.‬‬ ‫?‪What causes errors‬‬ ‫اﻟﺤﺴﺎب‬ ‫ ﺑﺎﻟﻨﺴﺒﺔ ﻟﻠﻤﺮﺑﻌﺎت اﻟﺼﻐﻴﺮة اﻟـ ‪25‬‬ ‫ ﻋﺪد اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ )ﻟﻜﻞ ﻣﻴﻜﺮوﻟﺘﺮ( = )ﻋﺪد اﻟﺨﻼﻳﺎ‬ ‫اﻟﻤﺤﺴﻮﺑﺔ ‪ /‬ﻋﺪد اﻟﻤﺮﺑﻌﺎت اﻟﻤﺤﺴﻮﺑﺔ( × اﻟﺘﺨﻔﻴﻒ × ‪.250‬‬ ‫ﻣﺎ اﻟﺬي ﻳﺴﺒﺐ اﻷﺧﻄﺎء؟‬  ‫ﻋﺪ ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء‬ :‫ ﻳﺘﻜﻮن ﻣﺤﻠﻮل ﻫﺎﻳﻢ ﻣﻤﺎ ﻳﻠﻲ‬ ‫ ﺟﺮام )ﻣﺤﻠﻮل ﻣﺘﺴﺎوي‬1 = ‫ ﻛﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم‬.1.(‫اﻟﺘﻮﺗﺮ‬.‫ ﺳﻴﻤﻨﻊ ﺗﻜﻮﻳﻦ اﻟﺮوﻟﻮ‬.‫ ﺟﺮام‬5 = ‫ ﻛﺒﺮﻳﺘﺎت اﻟﺼﻮدﻳﻮم‬.2.‫ ﺟﺮام ﻳﻌﻤﻞ ﻛﻤﻄﻬﺮ‬0.5 = ‫ ﻛﻠﻮرﻳﺪ اﻟﺰﺋﺒﻖ‬.3 ‫ ﻣﻞ‬200 = ‫ ﻣﺎء‬.‫ د‬.4 count RBCs Hayme’s solution consists of the following: 1.Na Cl = 1 G (Isotonic solution). 2.Na2SO4 = 5 grams. It will prevent rouleux formation. 3.HgCl2 = 0.5 G acts as an antiseptic. 4.D. H2O = 200 mL Procedure for RBC counting: 1. a dilution of 1:200 with a diluting solution. 2.Fill the red bulb pipette up to 0.5 marks with the blood. 3. Draw the solution to mark 101 of the RBC pipette. :‫إﺟﺮاء ﻋﺪ ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء‬.‫ ﺑﻤﺤﻠﻮل ﻣﺨﻔﻒ‬1:200 ‫ ﺗﺨﻔﻴﻒ‬.1.‫ ﻋﻼﻣﺔ ﺑﺎﻟﺪم‬0.5 ‫ اﻣﻸ اﻟﻤﺎﺻﺔ ذات اﻟﻠﻤﺒﺔ اﻟﺤﻤﺮاء ﺣﺘﻰ‬.2.‫ ﻋﻠﻰ ﻣﺎﺻﺔ ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء‬101 ‫ ارﺳﻢ اﻟﻤﺤﻠﻮل ﻟﻌﻼﻣﺔ‬.3  ‫ ﺑﺎﻟﻨﺴﺒﺔ ﻟﺨﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء‪ ،‬اﺳﺘﺨﺪم اﻟﻤﺮﺑﻊ اﻟﻤﺮﻛﺰي‪ ،‬اﻟﺬي ﻳﺤﺘﻮي‬ ‫ﻋﻠﻰ ‪ 25‬ﻣﺮﺑﻌً ﺎ أﺻﻐﺮ‪.‬‬ ‫ اﺣﺴﺐ ‪ 4‬ﻣﺮﺑﻌﺎت ﻓﻲ اﻟﺰاوﻳﺔ وﻣﺮﺑﻌً ﺎ ﻣﺮﻛﺰﻳًﺎ واﺣﺪً ا‪.‬‬ ‫ اﺣﺴﺐ ﻓﻘﻂ ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء اﻟﺘﻲ ﺗﻘﻊ ﻋﻠﻰ اﻟﺤﺪود اﻟﻴﺴﺮى‬ ‫واﻟﻌﻠﻮﻳﺔ ﻟﻬﺬه اﻟﻤﺮﺑﻌﺎت‪.‬‬ ‫‪.4‬ﻛﺮر اﻟﻌﺪ ﻣﺮﺗﻴﻦ واﻗﺴﻢ ﻋﻠﻰ ‪ 2‬ﻟﻠﺤﺼﻮل ﻋﻠﻰ اﻟﻤﺘﻮﺳﻂ‪.‬‬ ‫‪ For RBCs, use the center square, which has 25‬‬ ‫‪smaller squares.‬‬ ‫‪ Count the corner 4 squares and one central square.‬‬ ‫‪ Count only the RBCs that fall on these squares’ left‬‬ ‫‪and top borders.‬‬ ‫‪4.Repeat the count twice and divide by 2 to get the‬‬ ‫‪average.‬‬ ‫‪ The formula for RBCs count is as follows:‬‬ ‫‪1. Multiply factor = 10 x 200 / 0.2 = 10,000‬‬ ‫‪2. Multiply RBC count by 10,000 = RBCs million/cmm.‬‬ ‫ ﺻﻴﻐﺔ ﺣﺴﺎب ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء ﻫﻲ ﻛﻤﺎ ﻳﻠﻲ‪:‬‬ ‫‪.1‬اﺿﺮب اﻟﻌﺎﻣﻞ = ‪.10000 = 0.2 / 200 × 10‬‬ ‫‪.2‬اﺿﺮب ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺤﻤﺮاء ﻓﻲ ‪= 10000‬‬ ‫ﻣﻠﻴﻮن ﺧﻠﻴﺔ دم ﺣﻤﺮاء‪/‬ﻣﻠﻴﻤﺘﺮ ﻣﻜﻌﺐ‪.‬‬  ‫ﺿﻤﺎن اﻟﺠﻮدة‬ ‫ﻋﺪ اﻟﺨﻼﻳﺎ اﻟﻤﻜﺮرة‬.‫ ﻳﻤﻜﻦ اﺳﺘﺨﺪام ﻫﺬا ﻟﺠﻤﻴﻊ أﻧﻮاع ﻋﺪ اﻟﺨﻼﻳﺎ‬ ‫ ﻓﻴﺠﺐ أن ﻳﻜﻮن ﻫﻨﺎك‬،‫ إذا ﺗﻢ اﻟﻌﺪ ﻓﻲ ﻧﺴﺨﺘﻴﻦ ﻣﻜﺮرﺗﻴﻦ‬ Quality assurance ‫ ﻛﺤﺪ أﻗﺼﻰ ﻓﻲ اﻟﻨﺘﻴﺠﺔ اﻟﻨﻬﺎﺋﻴﺔ‬%20 ‫اﺧﺘﻼف ﺑﻨﺴﺒﺔ‬ Duplicate counting This can be used for all types of cell-counting. If counted in duplicate, there should be at maximum 20% difference in the final result Inter-square difference This works only for higher numbers of WBCs. In the 4 different squares counted, one should have a maximum of 10% difference. ‫اﻟﻔﺮق ﺑﻴﻦ اﻟﻤﺮﺑﻌﺎت‬.‫ ﻻ ﻳﻨﻄﺒﻖ ﻫﺬا إﻻ ﻋﻠﻰ اﻷﻋﺪاد اﻟﻜﺒﻴﺮة ﻣﻦ ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء‬ ‫ ﻳﺠﺐ أن‬،‫ ﻓﻲ اﻟﻤﺮﺑﻌﺎت اﻷرﺑﻌﺔ اﻟﻤﺨﺘﻠﻔﺔ اﻟﺘﻲ ﺗﻢ ﺣﺴﺎﺑﻬﺎ‬.%10 ‫ﻳﻜﻮن ﻫﻨﺎك ﻓﺮق ﺑﺤﺪ أﻗﺼﻰ‬  ‫إن ﺣﺴﺎب ﻋﺪد اﻟﺨﻼﻳﺎ ﻓﻲ ﺳﻮاﺋﻞ اﻟﺠﺴﻢ ﻳﻤﻜﻦ أن ﻳﺰودك ﺑﻤﻌﻠﻮﻣﺎت ﻣﻔﻴﺪة ﻟﻠﻐﺎﻳﺔ‪.‬‬ ‫‪Counting cells in body fluids can provide you with very useful‬‬ ‫‪information.‬‬ ‫‪automated cell-counters are used, but these have to:‬‬ ‫‪ be maintained properly, should be calibrated on a regular basis, and they‬‬ ‫‪need a steady supply of electricity.‬‬ ‫‪ Programming and technical interventions are also quite complex for these‬‬ ‫‪machines.‬‬ ‫‪ for example of a white blood cell (WBC) count in a cerebro-spinal fluid‬‬ ‫‪(CSF).‬‬ ‫ُﺗﺴﺘﺨﺪم ﻋﺪادات اﻟﺨﻼﻳﺎ اﻵﻟﻴﺔ‪ ،‬وﻟﻜﻦ ﻳﺠﺐ أن‪:‬‬ ‫ ﻳﺘﻢ ﺻﻴﺎﻧﺘﻬﺎ ﺑﺸﻜﻞ ﺻﺤﻴﺢ‪ ،‬وﻳﺠﺐ ﻣﻌﺎﻳﺮﺗﻬﺎ ﺑﺸﻜﻞ ﻣﻨﺘﻈﻢ‪ ،‬وﺗﺤﺘﺎج إﻟﻰ إﻣﺪاد ﺛﺎﺑﺖ ﻣﻦ اﻟﻜﻬﺮﺑﺎء‪.‬‬ ‫ ﺗﻌﺘﺒﺮ اﻟﺒﺮﻣﺠﺔ واﻟﺘﺪﺧﻼت اﻟﻔﻨﻴﺔ ﻣﻌﻘﺪة ﻟﻠﻐﺎﻳﺔ ﺑﺎﻟﻨﺴﺒﺔ ﻟﻬﺬه اﻵﻻت‪.‬‬ ‫ ﻋﻠﻰ ﺳﺒﻴﻞ اﻟﻤﺜﺎل ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء )‪ (WBC‬ﻓﻲ اﻟﺴﺎﺋﻞ اﻟﺪﻣﺎﻏﻲ اﻟﺸﻮﻛﻲ )‪.(CSF‬‬ ‫ﻏﺮﻓﺔ ﻋﺪ ﻓﻮﻛﺲ‪-‬روزﻧﺘﺎل‬ ‫اﻟﻤﺴﺎﺣﺔ‪ 4 :‬ﻣﻢ × ‪ 4‬ﻣﻢ‬ ‫اﻟﻌﻤﻖ‪ 0.2 :‬ﻣﻠﻢ‬ ‫اﻟﺤﺠﻢ اﻹﺟﻤﺎﻟﻲ‪ 3,2 :‬ﻣﻢ‪ ³‬أو ‪ul‬‬ ‫‪Fuchs-Rosenthal counting chamber‬‬ ‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﺴﺎﺋﻞ ‪ CSF‬ﻏﻴﺮ اﻟﻤﺨﻔﻒ‪ ،‬ﻗﻢ ﺑﻌﺪّ اﻟﺨﻼﻳﺎ‬ ‫ﻓﻲ اﻟﻤﺮﺑﻌﺎت ‪ 1‬و‪ 2‬و‪ 3‬و‪ 4‬و‪.5‬ﻋﺪد اﻟﺨﻼﻳﺎ‬ ‫اﻟﻤﺤﺴﻮﺑﺔ ﻓﻲ ﻫﺬه اﻟﻤﺮﺑﻌﺎت اﻟﺨﻤﺴﺔ ﻳﺴﺎوي ﻋﺪد‬ ‫اﻟﺨﻼﻳﺎ ﻟﻜﻞ ‪ μι‬ﻣﻦ ﺳﺎﺋﻞ ‪CSF‬‬ ‫ ﻏﺮﻓﺔ ﻋﺪ ﻣﺎﻻﺳﻴﺰ‬ ‫اﻟﻤﺴﺎﺣﺔ‪ 2 :‬ﻣﻢ × ‪ 2.5‬ﻣﻢ‬ ‫اﻟﻌﻤﻖ‪ 0.2 :‬ﻣﻠﻢ‬ ‫‪ Mallassez counting chamber‬‬ ‫اﻟﺤﺠﻢ اﻹﺟﻤﺎﻟﻲ ‪ 1‬ﻣﻢ‪ ³‬أو ‪ul‬‬ ‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﺴﺎﺋﻞ ‪ CSF‬ﻏﻴﺮ اﻟﻤﺨﻔﻒ‪ ،‬ﻓﺈن‬ ‫ﻋﺪد اﻟﺨﻼﻳﺎ اﻟﻤﺤﺴﻮﺑﺔ ﻓﻲ ﺣﺠﺮة اﻟﻌﺪ‬ ‫ﺑﺄﻛﻤﻠﻬﺎ ﻳﺴﺎوي ﻋﺪد اﻟﺨﻼﻳﺎ ﻟﻜﻞ‬ ‫ﻣﻴﻜﺮوﻟﺘﺮ ﻣﻦ ﺳﺎﺋﻞ ‪.CSF‬‬ ‫‪Kova (disposable) counting chamber‬‬ ‫ﺣﺠﺮة اﻟﻌﺪ ﻛﻮﻓﺎ )ﻟﻼﺳﺘﺨﺪام ﻣﺮة واﺣﺪة(‬ ‫ﺗﺤﺘﻮي ﺷﺮﻳﺤﺔ ﺑﻼﺳﺘﻴﻜﻴﺔ واﺣﺪة ﻋﻠﻰ ‪ 10‬ﺣﺠﺮات ﻋﺪ‪.‬ﺗﺘﻜﻮن اﻟﺤﺠﺮة‬ ‫ﺻﻐﻴﺮا‪.‬‬ ‫ً‬ ‫اﻟﻮاﺣﺪة ﻣﻦ ‪ 9‬ﻣﺮﺑﻌﺎت ﻛﺒﻴﺮة ﻣﻘﺴﻤﺔ إﻟﻰ ‪ 81‬ﻣﺮﺑﻌً ﺎ‬ ‫اﻟﺤﺠﻢ اﻹﺟﻤﺎﻟﻲ‪ul 1 :‬‬ ‫ﺣﺠﻢ اﻟﻤﺮﺑﻊ اﻟﺼﻐﻴﺮ ‪ 0,0123‬ﻣﻴﻜﺮوﻣﺘﺮ‬ ‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﺴﺎﺋﻞ ‪ CSF‬ﻏﻴﺮ اﻟﻤﺨﻔﻒ‪ ،‬ﻓﺈن ﻋﺪد اﻟﺨﻼﻳﺎ اﻟﻤﺤﺴﻮﺑﺔ ﻓﻲ ﺣﺠﺮة‬ ‫اﻟﻌﺪ ﺑﺄﻛﻤﻠﻬﺎ ﻳﺴﺎوي ﻋﺪد اﻟﺨﻼﻳﺎ ﻟﻜﻞ ﻣﻴﻜﺮوﻟﺘﺮ ﻣﻦ ﺳﺎﺋﻞ ‪CSF‬‬  ‫ أﻇﻬﺮ‬.‫ ﻳﺘﻢ ﺣﺴﺎب أرﺑﻌﺔ ﻣﺮﺑﻌﺎت زاوﻳﺔ ﻛﺒﻴﺮة‬.1/100 ‫ اﺣﺴﺐ ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء اﻟﺘﺎﻟﻲ إذا ﻛﺎن اﻟﺘﺨﻔﻴﻒ‬.1 :‫اﻟﺴﺆال‬ ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء‬25 = 3 ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء اﻟﻤﺮﺑﻊ‬28 = 2 ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء اﻟﻤﺮﺑﻊ‬30 = 1 ‫ اﻟﻤﺮﺑﻊ‬2 ‫ اﻟﺠﺎﻧﺐ‬1 ‫ اﻟﺠﺎﻧﺐ‬.‫ﺣﺴﺎﺑﺎﺗﻚ‬ = 3 ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء اﻟﻤﺮﺑﻊ‬23 = 2 ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء اﻟﻤﺮﺑﻊ‬27 = 1 ‫ ﺧﻠﻴﺔ دم ﺑﻴﻀﺎء اﻟﻤﺮﺑﻊ‬27 = 4 ‫اﻟﻤﺮﺑﻊ‬.‫ أﻇﻬﺮ ﺣﺴﺎﺑﺎﺗﻚ‬.‫ ﻳﺘﻢ ﺣﺴﺎب أرﺑﻌﺔ ﻣﺮﺑﻌﺎت زاوﻳﺔ ﻛﺒﻴﺮة‬.1/100 ‫ اﺣﺴﺐ ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء اﻟﺘﺎﻟﻲ إذا ﻛﺎن اﻟﺘﺨﻔﻴﻒ‬.1 Question: 1. Calculate the following white blood cell count if the dilution 1/100. Four large corner squares are counted. Show your calculations.Side 1Side 2Square 1 = 30 white blood cells Square 2 = 28 white blood cellsSquare 3 = 25 white blood cellsSquare 4 = 27 white blood cellsSquare 1 = 27 white blood cellsSquare 2 = 23 white blood cellsSquare 3 = 1. Calculate the following white blood cell count if the dilution 1/100. Four large corner squares are counted. Show your calculations. Side 1 Side 2 Square 1 = 30 white blood cells Square 1 = 27 white blood cells Square 2 = 28 white blood cells Square 2 = 23 white blood cells Square 3 = 25 white blood cells Square 3 = 24 white blood cells Square 4 = 27 white blood cells Square 4 = 32 white blood cells 2. Calculate the following white blood cell count if the dilution is 1/100. Four large corner squares are counted. Show your calculations. Total cells counted on one side = 36 Total cells counted on the other side = 32 3. Calculate the following white cell count if the dilution is 1 / 20, and all 9 large squares are counted. Please perform the calculation even though it is greater than 10%. Total cells counted on one side = 52 Total cells counted on the other side = 60 ‫ ﺗﻢ ﺣﺴﺎب أرﺑﻌﺔ‬.1/100 ‫ اﺣﺴﺐ ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء اﻟﺘﺎﻟﻲ إذا ﻛﺎن اﻟﺘﺨﻔﻴﻒ‬.2 36 = ‫ إﺟﻤﺎﻟﻲ اﻟﺨﻼﻳﺎ اﻟﻤﺤﺴﻮﺑﺔ ﻋﻠﻰ أﺣﺪ اﻟﺠﺎﻧﺒﻴﻦ‬.‫ أﻇﻬﺮ ﺣﺴﺎﺑﺎﺗﻚ‬.‫ﻣﺮﺑﻌﺎت زاوﻳﺔ ﻛﺒﻴﺮة‬ 32 = ‫إﺟﻤﺎﻟﻲ اﻟﺨﻼﻳﺎ اﻟﻤﺤﺴﻮﺑﺔ ﻋﻠﻰ اﻟﺠﺎﻧﺐ اﻵﺧﺮ‬ ‫ وﺗﻢ ﺣﺴﺎب ﺟﻤﻴﻊ‬،1/20 ‫ اﺣﺴﺐ ﻋﺪد ﺧﻼﻳﺎ اﻟﺪم اﻟﺒﻴﻀﺎء اﻟﺘﺎﻟﻲ إذا ﻛﺎن اﻟﺘﺨﻔﻴﻒ‬.3 ‫ إﺟﻤﺎﻟﻲ اﻟﺨﻼﻳﺎ‬.%10 ‫ ﻳﺮﺟﻰ إﺟﺮاء اﻟﺤﺴﺎب ﺣﺘﻰ ﻟﻮ ﻛﺎن أﻛﺒﺮ ﻣﻦ‬.‫اﻟﻤﺮﺑﻌﺎت اﻟﻜﺒﻴﺮة اﻟﺘﺴﻌﺔ‬ 60 = ‫ إﺟﻤﺎﻟﻲ اﻟﺨﻼﻳﺎ اﻟﻤﺤﺴﻮﺑﺔ ﻋﻠﻰ اﻟﺠﺎﻧﺐ اﻵﺧﺮ‬52 = ‫اﻟﻤﺤﺴﻮﺑﺔ ﻋﻠﻰ أﺣﺪ اﻟﺠﺎﻧﺒﻴﻦ‬