Lecture 11 Flashcards
Hemocytometer or Counting Chamber
- Glass ‘chamber’ sits on microscope stage
- Grid area- Neubauer chamber (etched into glass) to count cells
- Special thick coverslip holds dilution in place
- Creates a well of 0.1mm depth
- Two sides – count cells both sides and average counts
- System reusable
*Charge with10µL of the fluid or dilution using an
Eppendorf pipette, hold at 45°while touching the tip to the Sample Injection Port.
-Each large square is 1 mm made up of 25 small squares 0.25 mm and lines of 0.05 mm
-Count 4 large corner squares for WBC
‒Count 5 small central squares- 4 small corner
squares and 1 small middle square =- RBC - PLTs - count entire center square (outlined in blue- 25 small squares)
C-Chip Counting Chamber - single use plastic with Two ‘half-moon’ ports - sample injection or ‘charging’ stations
Dilutions for Manual Cell CountsHemocytometer
- RBC – 1:10 up to 1:200 dilution in isotonic saline – keeps RBCs intact – high dilution
- WBC – 1:20 dilution using weak diluting fluid:
‒Acetic acid or Buffered Ammonium oxalate
‒RBCs are lysed, WBC and PLT left intact only count WBC - PLT – 1:100 dilution in 1% Ammonium oxalate
‒RBC lysed, WBC & PLT left intact - Count PLTs based on size and morphology
‒Adjust dilution if platelets too few or too numerous
Add dye and reduce light to help visualize
-Only Count cells on top & left of grid lines two slides should not differ more than 10%
Source of Error for \Hemocytometer
Improperly prepared dilution
Dirty chamber –
Air bubbles or dirt/debris
Incorrectly set–up microscope: Can’t see the cells
Improperly MIXED dilution – Leads to uneven distribution of cells
Improperly prepared dilution -Falsely high or low count
Dirty chamber with Air bubbles or dirt/debris-Falsely high count
Incorrectly set–up microscope: Can’t see the cells -Falsely low count
Improperly MIXED dilution Leads to uneven distribution of cells -Falsely low count
Source of Error: Hemocytometer
Over-filling chamber – Cells lost in moats/troughs
Under-filling chamber – Less cells, drying out faster
Not following SOP timing – Cells do not settle before counting
Not following SOP counting rules – Counting cells twice
Calculation errors Incorrect formula applied
Incorrect Reporting - Transcription error(s)
Over-filling chamber – Cells lost in moats/troughs
-Falsely low count
Under-filling chamber – Less cells, drying out faster
-Falsely low count
Not following SOP timing – Cells do not settle before counting –Falsely low count
Not following SOP counting rules – Counting cells twice -Falsely high count
Calculation errors Incorrect formula applied -Falsely high or low count
Incorrect Reporting - Transcription error(s)
Wrong result(s)
Hematocrit (HCT)
-volume of packed RBCs in whole blood
* Also known as Packed Cell Volume (PCV)
* % or in L/L
* Perform manual HCT when WBC is ↑ ↑ ↑ and sample requires dilution
* Use with automated HGB to calculate RBC indices
Microhematocrit (or Manual HCT)
- determined by spinning a blood filled capillary tube in a centrifuge
- EDTA or heparinized capillary tubes (red) regular (blue) and ends are sealed with clay
*fill two capillaries of EDTA blood, wipe, seal, centrifuge at 10000
*read RBC packing not the buffy coat. Convert L/L
-there will be three layers- Plasma, buffy cota and erythrocytes
Sources of Error Microhematocrit (or Manual HCT
- Improperly collected specimen (incorrect venipuncture or finger-prick technique)
Inadequate mixing of blood
An increased amount of anticoagulant (incorrect type, amount, or concentration of anticoagulant)
Improper sealing of the capillary tube
Improperly collected specimen (incorrect venipuncture or finger-prick technique)
- presence of interstitial fluid causes decreased HCT readings
Inadequate mixing of blood
-Can cause false increases or decreases in HCT due to clotting
An increased amount of anticoagulant (incorrect type, amount, or concentration of anticoagulant)
-Decreases the HCT reading as a result of erythrocyte shrinkage
Improper sealing of the capillary tube
Causes a decreased HCT reading as a result of loss of blood during centrifugation
Sources of Error Microhematocrit (or Manual HCT
insufficient centrifugation (time and speed)
Use of brake during centrifugation
Delay between centrifugation and interpretation of results:
Improper use of the micro-hematocrit reader/improper reading of HCT tube
Certain disorders may cause plasma to be trapped in the RBC layer (Wintrobe method):
insufficient centrifugation (time and speed)
-‒HCT falsely increased or decreased
Use of brake during centrifugation
-RBC layer may be forced forward and result in a falsely elevated result
Delay between centrifugation and interpretation of results: RBCs begin to settle resulting in a false HCT reading
Improper use of the micro-hematocrit reader/improper reading of HCT tube
-‒Inclusion of the buffy coat in HCT reading falsely elevates the result
Certain disorders may cause plasma to be trapped in the RBC layer (Wintrobe method):
‒ Sickle cell anemia
‒ Macrocytic anemia’s
‒ Hypochromic anemia’s
‒ Spherocytosis
‒ Thalassemia
A temporarily low HCT reading may result immediately after blood loss, as plasma is replaced faster than erythrocytes
The main measurements of RBCs include
‒ Total number of RBC in one liter of blood or RBC count in x 1012 / L
‒ Total hemoglobin content in one liter of blood or HGB in g/L
‒ Total volume of all RBCs within the whole blood or Hematocrit (HCT) in L/L
Mean Cell Volume
(MCV)
The average size or volume of an
RBC (fL)
80 – 100 fL
HCT x 1000/ RBC
Mean Cell Hemoglobin
(MCH)
The average weight or mass of
Hemoglobin per RBC (pg)
26 - 34 pg
HGB/RBC
Mean Cell Hemoglobin
Concentration
(MCHC)
The average concentration of
Hemoglobin per RBC (g/L)
320 – 360 g/L
HGB/HCT
RBC Distribution Width
(RDW)
The variation in size of all of the
RBCs (expressed as %)
11.5 - 14.5
Taken from RBC Histogram on CBC report
Hemoglobin Measurement
-done using Cyanmethemoglobin Method
* Lyze the RBC to free the HGB into supernatant
* [K3Fe(CN)6] converts Hgb to Methemoglobin, iron is oxidized to ferric
-KCN converts Methemoglobin to Cyanmethemoglobin
-* Blood is mixed with Drabkin’s reagent
‒20µL Whole EDTA blood to 5.0mL reagent
- Drabkin’s reagent contains:
‒ Potassium ferricyanide [K3Fe(CN)6]
‒ Potassium cyanide [KCN]
‒ Sodium bicarbonate
‒ Surfactant
-measured by dilution reading on spectrophotometer at 540 nm with Drabkins reagent as blank. Plot a standard curve
- Cyanmethhemoglobin Absorbance is directly proportional to Hgb concentration
Cyanmethemoglobin Method –
Sources of Error
- Reagent sensitive to light
- Reagent contains cyanide
- Spectrophotometers require calibration
- Elevated WBC or PLT counts, lipemia, HGB S and C crystals
- Abnormal globulins (
- Reagent sensitive to light
‒ Store in brown bottle or away from light - Reagent contains cyanide – handle with care, including appropriate waste disposal
- Spectrophotometers require calibration
‒ Commercially prepared absorbance standard kits are available - Elevated WBC or PLT counts, lipemia, HGB S and C crystals cause turbidity and falsely high results
- Abnormal globulins (plasma cell myeloma or Waldenstrom macroglobulinemia) can precipitate in the reagent
‒ Reagent modified to contain KH2 PO4 salt which eradicates the problem
