Lecture 20 Manual Methods

Question 1

Zeta Potential

Answer 1

• net negative charge on RBC membrane due to sialic acid present

Question 2

Erythrocyte Sedimentation Rate

Answer 2

• distance in mm that the RBCs fall in 1 hr

- increased ESR due to macrocytes or formation of rouleaux

Question 3

ESR Normal Ranges

Answer 3

Male (Westergren):
- 0-15 mm/hr (0-50 yrs old)
- 0-20 mm/hr (>50 yrs old)
Female (Westergren):
- 0-20 mm/hr (0-50 yrs old)
- 0-30 mm/hr (>50 yrs old)

Question 4

Stages of Sedimentation

Answer 4

1. Lag phase: first few minutes rouleaux occurs and aggregates form
2. Decantation phase: sinking or settling of red cells at a relatively constant speed
3. Packing phase: sedimentation slows as cells pack at bottom of tube

Question 5

Falsely Increased ESR

Answer 5

• tilted pipette tube
• test time >1 hr
• improper dilution
• improper mixing
• increased temp (>25 deg C)
• vibration
• sodium or potassium oxalate anticoagulants, heparin make RBCs shrink, fall faster

Question 6

Falsely Decreased ESR

Answer 6

• refrigerated sample
• sample > 4hrs old
• excess anticoagulant
• test time < 1 hr
• bubbles in ESR column and clotted sample
• improper blood dilution
• improper mixing
• cool room temp

Question 7

Increased results (clinical conditions)

Answer 7

• pregnancy
• anemia
• macrocytosis
• inflammatory disease (rheumatoid arthritis)
• cancer
• acute/chronic infections
• increased plasma globulins (ie multiple myeloma)
• increased plasma fibrinogen
• tuberculosis
• smokers

Question 8

Decreased results (clinical conditions)

Answer 8

• presence of sickle cells
• polycythemia
• spherocytosis
• microcytosis
• hypofibrinogenemia
• increased plasma viscosity

Question 9

ESR Normal

Answer 9

• osteoarthritis
• first 24 hrs of acute appendicitis
• viral infection
• angina pectoris

Question 10

ESR Elevated

Answer 10

• rheumatic fever
• early stage of acute pelvic inflammatory disease
• bacterial infection
• myocardial infarction

Question 11

Microhematocrit

Answer 11

• hematocrit is the volume of packed cells that occupies a given volume of whole blood
• sometimes referred to as packed cell volume (PCV)

Question 12

Microhematocrit Layers

Answer 12

1. Plasma
2. Platelets (white layer)
3. Leukocytes
4. Reticulocytes and nucleated red cells
5. Mature red cells
6. Clay seal

Question 13

Microhematocrit Sources of Error

Answer 13

• improper sealing
• increased conc of anticoagulant -> rBC shrinkage, falsely decr. results
• incorrect use of microhematocrit reader
• time and speed of centrifugation
• buffy coat should not be included in the reading
• disorders with abnormal RBC (ie sickle cell anemia, spherocytosis, thalassemias)
• a temp low Hct may result after blood loss as plasma replaced faster than RBCs
• dehydration causes low plasma volume -> falsely hi Hct values
• addition of fluid from venipuncture insterstitial sources and improper flushing of lines -> falsely decr values

Question 14

Hct normal ranges

Answer 14

Male:
- 40-54%
- 0.40-0.54 L/L
Female:
- 35-49%
- 0.35-0.49 L/L

Question 15

Hemoglobin Measurement

Answer 15

• cyanmethemoglobin (ref method)
• lysing reagent frees Hgb from RBCs
• free Hgb combines with potassium ferricyanide
• converts Hb iron from ferrous->ferric (forms methemoglobin)
• all forms of hemoglobin converted to methemoglobin except sulfhemoglobin
• pigment cyanmethemoglobin read spectrophotometrically

Question 16

Hgb Normal Ranges

Answer 16

Adult Male:
- 135-180 g/L
Adult Female:
- 120-150 g/L

Question 17

Alternate Hgb measurement

Answer 17

POC:
- azide methemoglobin
- benefit: no dilution; turbidity not a factor
Sysmex:
- sodium lauryl sulfate
- benefit: not toxic waste

Question 18

Hgb Sources of Error

Answer 18

• related to procedure
• related to specimen (ie lipemia, icteric)
• related to Pt condition (leukocytosis)

Question 19

Retic Count Ref Range

Answer 19

• newborn values are high
  Adult:
• 0.5-2.5%
• 20-115 x 10^9/L

Question 20

Retic Count

Answer 20

• retics appear after application of new methylene blue
• 2 or more granules
• to determine if BM capable for producing RBCs in response to anemia

Question 21

Retic Count Sources of Error

Answer 21

• adjust proportion of blood:
  
  • severe anemia (incr blood, decr stain)
  • polycythemic (decr blood or incr stain)
• mix well
• avoid humidity, poor drying -> refractile appearance
• other cell inclusions also stain supravitally: Heinz bodies, Howell-Jolly, Pappenheimer, Basophilic Stippling, Hgb H inclusions, Cabot Rings

Question 22

Retic Calculation

Answer 22

% Retic = retic count/1000 RBCs/10

Absolute Retic Count = retic % x RBC count (x10^12/L)/100

Question 23

Automated Retic Counts

Answer 23

• now offered on automated analyzers
• based on principle of optical scatter or fluorescence
• % and absolute counts provided

Question 24

Mean Cell Volume (MCV)

Answer 24

MCV = Hct (L/L) x 10^3/L/ RBC (x10^12/L)

Question 25

Mean Cell Hemoglobin (MCH)

Answer 25

MCH = Hgb (g/L)/ RBC (x10^12/L)

Question 26

MCHC (Mean Cell Hemoglobin Conc.)

Answer 26

MCHC = Hgb (g/L)/ Hct (L/L)

Question 27

Buffy Coat Prep

Answer 27

1. Centrifuge EDTA anticoagulated whole blood in plastic tube
2. Remove only plasma above buffy coat using fine pipette and transfer to fresh tube
3. Use same pipette to remove buffy coat onto1 or 2 slides
4. Mix with one drop of patient plasma and prepare PBS

Question 28

Buffy Coat Use

Answer 28

• detection of small numbers of abnormal cells
• concentrate WBCs when there is low WBC count
• detection of bacteria/fungi/parasites