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LO1 Flashcards

1
Q

Neubauer Hemocytometer

-area of the entire cytometer including units
-what is the total volume
-The depth of the ruled area is
-is the whole cytometer usually counted?

A

-Area - All 9 squares = 9mm^2
-Total Volume = 0.9 mm^3 = 0.9 uL
-depth is 0.1 mm
-yes usually all counted

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2
Q

what kind of slips are used form the
Improved Neubauer Hemacytometer

A

An optically flat, calibrated coverslip
made from quartz

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3
Q

the principle of manual cell count steps ish

A

-mix the specimen first
-specimen is then flooded onto a hemocytometer
-the cellular elements are counted microscopically and reported

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4
Q

what is the most common specimen used on the hemocytometer
1-4

A

CSF
Body fluids – second most common
Joint fluids, paracentesis, dialysis fluids
Sperm samples

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5
Q

To prepare for flooding:
-clean what with what
-dry with what

A
  • Clean the hemocytometer and coverslip with distilled water followed by alcohol.
  • Dry thoroughly with lint free paper so particles will not interfere with counting. Do not use gauze as it can scratch the chamber
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6
Q

actually flooding the chamber

A

-Place the coverslip on the hemocytometer
so that it partially covers both V slashes.

-Gently mix sample before flooding

-The area on the chamber must be filled but
not overfilled

-When flooded,the chamber must sit for
about 5 min to allow the cells to settle. This is
done in a wet place (a petri dish with wet
gauze can be used).

-want the cells to be well distributed

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7
Q

when counting all 9 squares what are excluded

A

the right edge and bottom edge

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8
Q

calculation review for the number of cells

A
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9
Q

Quality Control Check

distribution
undiluted vs diluted samples % checks

A

-Distribution checks – visually check

-If a fluid is undiluted, the duplicate counts must check within ± 25%.*

-If a dilution is required, duplicate counts must agree within ± 10%.*

-*This may vary between labs and may be based on actual number of cells
counted

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10
Q

CSFs are diluted with what

A

with saline because we do not want to lyse any cells.

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11
Q

Most common “fluid other than blood” analyzed in
Hematology is called

A

Cerebrospinal Fluid

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12
Q

Cerebrospinal Fluid some important points
begin to lyse within x hrs
maximam storage x hours at what temps
what must you do when sample arrives first
which number of tubes goes to hematology
what should we know range wise

A
  • Cells begin to lyze within 1 hour of collection.
  • Maximum storage is for 4 hours at 4 ̊C

-Must record specimen arrival time in lab

-Hematology – 3rd or last tube
- Critical range = > normal range

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13
Q

what are the 2 things/ qualities that must be reported on a CSF that we can see

A

colour & clarity

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14
Q

what is the normal colour & clarity of a CSF

A

clear and colorless

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15
Q

abnormal CSF
-color- special name + 4 other common colors
and the clarity

A

-Xanthochromia (yellow)
Pink , Red , Brown

-Cloudy

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16
Q

CSF Xanthochromia (yellow) means what :3 possible

A
  • Blood in the CSF >2-3 hr = usually hemorrhage
  • Liver disease
  • Presence of bilirubin, carotene and melanin
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17
Q

Pink , Red , Brown CSF

A

– Hemorrhage or traumatic tap?

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18
Q

cloudy CSF indicates what

A

Infection - WBCs

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19
Q

which side of the hemocyteometer is flooded for undiluted sample?
and which squares are counted at what power
what is being counted
how are calculations done

A

both sides, all 9 suares at 40X
RBC and WBC are counted
-calculations use avrg of both sides

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20
Q

CSF dilution and counts specifications

A

CSF is usually undiluted and the duplicate counts must check within ±25%. The specimen MAY be diluted with isotonic saline (0.85%). If diluted, duplicate counts must agree within ±10%

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21
Q

white vs red blood cells look

A

white are busier with things in middle, red it more donut like with a lighter middle and dark circle around

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22
Q

white and red blood cells Normal Reference Range - ADULT

A

RBC – 0 x 106/L – all ages
WBC – 0 – 5 x 106/L

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23
Q

Critical counts are counts that are

A

HIGHER than the normal reference range, and MUST be phoned to the ward/physician

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24
Q

Meningitis what is it :
caused by:
characterized by:

A

-Inflammation of membrane of the brain (meninges)

-caused by a bacterial or viral infection and

-characterized by high fever, severe headache, and stiff neck or back muscles.

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25
Increased lymphocytes suggest
viral or fungal meningitis
26
Neutrophils suggest
bacterial meningitis u Malignant cells may also be present
27
meninges of the CNS layers
skin periosteum bone dura matter arachnoid pia mater
28
Automated cell counts on CSF what is it (type of microscopy) and how does it compare to manual
Automated cell counters concentrate and enumerates RBC’s and WBC’s using fluorescent microscopy Manual chamber counts are still considered the “gold standard"
29
Procedure of manual platelet counting what is diluted and with what flooding which square is counted and what is the dilution of it how are the counts verified is it done in Sask?
- Whole blood is diluted with 1% ammonium oxalate. This diluent lyses the RBCs. - Flooding the hemocytometer is the same. - Different area counted and dilution factor – center primary square and 1/100 dilution. - Manual platelet counts are always verified by a peripheral blood smear estimate. These must check within 25%. - Not done in Saskatchewan anymore – but maybe elsewhere
30
platelet count calculation in book reiew
31
Platelet Reference range
150 – 450 x 10^9/L
32
Thrombocytopenia
defined as a platelet count less than 150 x 109/L
33
Thrombocytosis
defined as a platelet count greater than 450 x 10 9/L
34
Synovial Fluid
joint
35
Serous Fluid 2 types
- Pleural fluid – Thoracic - Peritoneal Fluid – Abdominal
36
can Eosinophils in Urine or Nasal discharge be found in those places
yes
37
ESR=
ERYTHROCYTE SEDIMENTATION RATE
38
ESR is a measure of
the distance (millimeters) that erythrocytes settle in the anti-coagulated plasma of a whole-blood specimen during a specified period of time (one hour). - Screening test – relatively non-specific.
39
3 Principle – Stages of the ESR
Lag phase - is the initial settling (~ 5 - 10 min) Rapid fall - constant rate of fall (~ 40 min) Packing phase - slower rate of fall (~10 min). Really only significant when discussing manual methods.
40
ESR usefullness
-used as a screening test for inflammatory response -monitoring therapy in autoimmune diseases (temporal arteritis, polymyalgia rheumatica and rheumatoid arthritis). In auto-immune diseases the ESR may exceed 100 mm/hour. -Temporal arteritis: chronic inflammation in large arteries -Polymyalgia rheumatica: rheumatic illness related to temporal arteritis
41
Zeta Potential
Net negative charge on the Red Blood Cell membrane which causes the RBC’s to repel one another as they move through the vascular system, and when they are suspended in plasma in a whole blood specimen. - Any changes in the zeta potential affect the RBC’s ability to rouleaux (stack ontop of one another), and therefore affect the ESR.
42
3 RBC factors that affect the ESR
u Shape – Can the cells rouleaux? u Number – Anemia, polycythemia? u Patient’s general health – Inflammation?
43
2: Enhance Rouleaux (Increased ESR)
u Increased plasma proteins (especially fibrinogen, beta and alpha globulins) u Decreased RBC numbers (decreased HCT)
44
Inhibit Rouleaux (Decreased ESR)
u Abnormal RBC shapes u spherocytes u sickle cells u Increased RBC numbers (increased HCT) u Increased plasma viscosity u Increased plasma albumin
45
What does rouleaux look like?
u The stacking up of blood cells one on top of the other, like pennies. u When they are stacked, they can then begin the rapid fall phase. u Anything that interferes with rouleaux formation will decrease the ESR. u Anything that enhances the formation of rouleaux will increase the ESR.
46
Pathological Conditions That Enhance Affect Rouleaux
Inflammation - appendicitis - pancreatitis Immunoglobulin disorders -Multiple Myeloma -Waldenstrom’s Macroglobulinemia Decreased RBC disorders - Iron deficiency anemia
47
Pathological Conditions That Inhibit Affect Rouleaux
Increased RBC’s -Polycythemia Vera -Burns -Dehydrations Abnormal RBC shapes -sickle cell anemia - spherocytosis
48
2 Physiological Conditions That Affect Rouleaux
* Pregnancy * Menstruation
49
The Test Method (just know there are diff) and Equipment for ESR
u Westergren ESR u Wintrobe ESR u Modified Westergren Equipment u ESR tube u ESR Rack u Timer capable of timing 1 hou
50
ESR Reference Ranges
u Males: 0 - 15 mm in one hour. u Females: 0 - 20 mm in one hour. u Children: 0 - 15 mm in one hour
51
Technical/Mechanical Factors (Sources of Error) affecting an ESR Tube position
u Tilt (3 degrees = 30% error) u Vibration = Increases ESR u Length and diameter of tube
52
Technical/Mechanical Factors (Sources of Error) affecting an ESR Temp
- Increased temperature = Increased ESR - Decreased temperature = Decreased ESR
53
Technical/Mechanical Factors (Sources of Error) affecting an ESR Time
: >4 hours @ RT or >24 hours @ 4 degrees = Decreases the ESR
54
Technical/Mechanical Factors (Sources of Error) affecting an ESR 3 others
- Anticoagulant used - over-coagulation (too much anticoagulant) - Proper mixing of specimen - Presence of anemia – may be seen as pathological or physiological
55
Quality Control of ESR
- Very few commercial controls available - Only manual test not run in duplicate -Quality of results rests with consistent, good technique and equipment to control sources of error. - ESR is a screening test only (not specific or precise). - If the ESR is normal, disease is not necessarily ruled out.
56
Label tubes for ESR
1. 3 identifiers Name (LAST NAME, first name) PHN Date of birth 2. Date and Time of Collection 3. Test ordered 4. Lab accession number
57
Reports
patient name correct? patient identification correct? result correct? reference range included? critical value phoned? for CSF everything is ciriticla, ESR has no critical Report signed and dated?
58
Recap qustions to think
- Define ESR - Define Zeta potential - Methodology - Sources of error
59
Review from LO 1
* When counting an undiluted CSF, how close must your duplicate counts be? * What about a diluted sample? * What are the units we report CSFs in? * What constitutes a “critical” result for a CSF? * What is the name of the condition with decreased platelets? Increased platelets? * What is zeta potential?
60
ESR Review
1. How long at room temp? 2. What would happen to ESR results if we forgot to turn the fan off in the biosafety cabinet? 3. How would spherocytes affect ESR? * Spherocytes inhibit rouleaux, falsely decreasing ESR.
61
Reporting CSF Colour and Clarity: Review
Color: * Colourless * Yellow * Pink * Red * Brown Clarity: * Clear * Slightly Cloudy * Cloudy
62
Hemoglobin . . . characterics
* Is a protein * Contains iron * Transports oxygen * Comprises ~ 35% of the cell volume * Is what makes blood red
63
The function of Hemoglobin is to . . 2
* Transport oxygen from the lungs to the body * Maintain body pH (pH inversely proportional to CO2)
64
heme essential component
Ferrous (+2) “Us” - carries oxygen
65
storage iron heme
Ferritin (+3) Ferric state = “Icky” – can not carry oxygen
66
HEME portion of Hemoglobin 2 prts
*- One Atom of Ferrous Iron *- One Mole of Oxygen
67
Globin portion of Hemoglobin
* Globin - has 4 amino acid chains
68
* 3 Different types of Hemoglobin present in a normal individual:
97% - Hemoglobin A (2 alpha/2 beta chains) 3% - Hemoglobin A2 (2 alpha/2 delta chains) <1% - Hemoglobin F (2 alpha/2 gamma chains)
69
Hemoglobin (HGB) evolution controlled by 2
* Evolves: controlled by genetics and gestational age
70
time and amounts of Embryonic hemoglobin → fetal hemoglobin → adult hemoglobin
* 30 weeks gestation: 90% to 95% Hemoglobin F * At birth: 53% to 95% Hemoglobin F * 12 weeks old: 7% Hemoglobin F * 6 months old: 2% to 3% Hemoglobin F
71
3 steps in Measuring Hemoglobin
Release the hemoglobin from the RBC Measure the hemolysate using a spectrophotometer Hemoglobin is a STABLE colored product
72
Principle of Spectophotometry 100% transmitted light = 0 =
100% transmitted light = 0% absorbance 0 = Blank made of diluent and calibrated to read 100% transmittance (0% absorbance) Measure unknown samples against the calibration curve This methodology is used in both manual and automated analysers
73
Cyanmethemoglobin Method what type of blood is used this blood is mixed with reagent containing 2 things it converts ferrous hemoglobin to what the absorbance is comapred to what all forms of hemoglobin is converted except which companied have developed what other reagents
Whole blood is mixed with a reagent containing potassium ferricyanide and potassium cyanide (Drabkins reagent) to convert ferrous hemoglobin to a very stable compound called cyanmethemoglobin (Fe+3) The absorbance peak of this cyanmethemoglobin can be measured at 540nm. The absorbance of the unknown sample is compared to a reference standard solution All forms of hemoglobin will be converted to cyanmethemoglbin except sulfhemoglobin. Companies have developed “non-cyanide” reagents. The principle remains the same. The wavelength will change slightly depending on the reagent components.
74
Sodium Lauryl Sulfate Method what is SLS max absorbance of SLS does it fallow beer law 2 advantages of SLS
* SLS is a surfactant which lyses RBC’s and forms a complex with the hemoglobin. * SLS-Methhemoglobin is stable and has a maximum absorbance of 539 nm. * ALSO follows Beer’s Law – precise linear correlation * Two advantages: less toxic, and less prone to interference from lipemia and increased WBC’s
75
4 prts of a Hemoglobin Curve units follows what law samples at what absorbance what are we reading off the graph?
Prepare a standard graph with dilutions of a certified standard. Follows Beer’s Law Measure prepared samples at 540 nm on a spectrophotometer Read concentration off the prepared graph
76
sample type for hemoglobin
* Whole Blood * EDTA * Heparinized capillary
77
hemoglobin Sources of Interference
* Turbidity will elevate a hemoglobin value * Patient conditions that cause turbidity: * Lipemia * Elevated WBC count > 100 x109/L * Elevated Platelet count > 700 x109/L * Hemoglobin variants that resist hemolysis (S or C) * Many analyzers incorporate a lysing agent that will lyse resistant RBC’s - more about this in LO3 * Carboxyhemoglobin – degree of error is not clinically significant
78
Reference Ranges hemoglobin
Males 135 - 180 g/L Females 115 - 164 g/L Newborns 160 - 240 g/L Why are males higher than females? Why are babies higher than adults?
79
MICROHEMATOCRIT PRINCIPLE hemocrit defined as
* Hematocrit (HCT) - defined as the volume of RBCs in a given volume of whole blood, expressed as litres of erythrocytes per litre of whole blood (L/L). * Microhematocrit - a small amount of whole blood is centrifuged to determine maximum packing of RBC expressed as HCT. * HCT is sometimes called Packed Cell Volume or PCV
80
microhematocrit The Method Equipment and Procedure
Capillary HCT tubes Sealing clay Centrifuge Reading device - % (USA)/ Canada (L/L) Example: * 45% = 45.0 mL RBC/100 mL blood * = 0.450 L RBC/1 L whole blood * = 0.450 L/L
81
Manual Hematocrit Microhematocrit method calculation
RBC volume in mm / =20.0 mm/ Total volume in mm 59.0 mm 0.33898 L/ 1L = 0.339 L/L
82
Technical Sources of Error for Manual Method hematocrit
Centrifuge: Time and speed must be constant Excess anticoagulant: RBCs shrink = decreased HCT Hemolysis: RBCs lysed = decreased HCT Improperly mixed specimen Capillary tubes not sealed: Leaks (all kinds of problems) Bubbles in tube Inaccurate readings
83
Physiological Sources of Error microhematocrit
* Trapped plasma – Automated hematology analyzers calculate the Hematocrit, therefore their results are not affected by trapped plasma and are lower. * Abnormal RBC shapes. * RBC shape may affect results. * Example: Spherocytes and sickle cells will falsely increase the microhematocrit because of incomplete packing.
84
Controlling Sources of Error microhematocrit
* Duplicate testing * Duplicates must agree within 0.02 L/L. * If duplicates do not agree, repeat the prodecure with newly mixed sample. * Rerun the sample in duplicate again. * Run a control with each run. * Controls are run in duplicate, like the patient. * Checks that the centrifuge speed and time are correct, and that the results are recorded accurately from the microhematocrit reader.
85
hematocrit Reference Ranges Remember – this is a test that NOW has three decimals when reporting
-Males 0.400 - 0.540 L/ -Females 0.330 - 0.480 L/L -Newborns 0.400 - 0.620 L/L
86
Clinical Significance Increased in certain disease states:
* Polycythemia * Dehydration - burn victims, severe diarrhea, vomiting
87
Clinical Significance decreased in certain disease states:
* Anemias * Leukemias * Bleeding disorders
88
clinical significant Hemorrhage - no change
Hemorrhage - no change for up to 24 hours, then plasma volume begins to increase to replace fluid loss and dilution of blood occurs
89
Specimen type microhematocrit 2 options
* Good specimen/collection: * No hemolysis * No tissue fluid * EDTA * Heparin * Capillary (plain or heparin)
90
Why EDTA in Hematology?
* Blood coagulation is inhibited by chelating, or binding, calcium ions. * Preserves cellular morphology for blood smears (should be made within 2 hours of collection). * Prevents platelet aggregation, therefore is preferred for platelet counts
91
Heparin in Hematology?
* Inactivates thrombin. * Morphological distortion of platelets & WBCs. * Causes bluish discoloration on blood films stained with a Romanowsky stain (because of its pH). * Can be used for the Microhematocrit and Hemoglobin ONLY

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