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hema > LO1 > Flashcards

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
Q

Increased lymphocytes suggest

A

viral or fungal meningitis

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26
Q

Neutrophils suggest

A

bacterial meningitis
u Malignant cells may also be present

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27
Q

meninges of the CNS layers

A

skin
periosteum
bone
dura matter
arachnoid
pia mater

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28
Q

Automated cell counts on CSF what is it (type of microscopy) and how does it compare to manual

A

Automated cell counters concentrate and
enumerates RBC’s and WBC’s using fluorescent
microscopy

Manual chamber counts are still considered the “gold
standard”

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29
Q

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?

A
  • 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
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30
Q

platelet count calculation in book reiew

A
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31
Q

Platelet Reference range

A

150 – 450 x 10^9/L

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32
Q

Thrombocytopenia

A

defined as a platelet count less than
150 x 109/L

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33
Q

Thrombocytosis

A

defined as a platelet count greater than
450 x 10 9/L

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34
Q

Synovial Fluid

A

joint

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35
Q

Serous Fluid 2 types

A
  • Pleural fluid – Thoracic
  • Peritoneal Fluid – Abdominal
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36
Q

can Eosinophils in Urine or Nasal discharge be found in those places

A

yes

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37
Q

ESR=

A

ERYTHROCYTE SEDIMENTATION RATE

38
Q

ESR is a measure of

A

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
Q

3 Principle – Stages of the ESR

A

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
Q

ESR usefullness

A

-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
Q

Zeta Potential

A

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
Q

3 RBC factors that affect the ESR

A

u Shape – Can the cells rouleaux?
u Number – Anemia, polycythemia?
u Patient’s general health – Inflammation?

43
Q

2: Enhance Rouleaux (Increased ESR)

A

u Increased plasma proteins (especially
fibrinogen, beta and alpha globulins)

u Decreased RBC numbers (decreased HCT)

44
Q

Inhibit Rouleaux (Decreased ESR)

A

u Abnormal RBC shapes
u spherocytes
u sickle cells
u Increased RBC numbers
(increased HCT)
u Increased plasma viscosity
u Increased plasma albumin

45
Q

What does rouleaux look like?

A

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
Q

Pathological Conditions That Enhance
Affect Rouleaux

A

Inflammation
- appendicitis
- pancreatitis
Immunoglobulin disorders
-Multiple Myeloma
-Waldenstrom’s Macroglobulinemia
Decreased RBC disorders
- Iron deficiency anemia

47
Q

Pathological Conditions That Inhibit
Affect Rouleaux

A

Increased RBC’s
-Polycythemia Vera
-Burns
-Dehydrations
Abnormal RBC shapes
-sickle cell anemia
- spherocytosis

48
Q

2 Physiological Conditions
That Affect Rouleaux

A
  • Pregnancy
  • Menstruation
49
Q

The Test Method (just know there are diff) and
Equipment for ESR

A

u Westergren ESR
u Wintrobe ESR
u Modified Westergren

Equipment
u ESR tube
u ESR Rack
u Timer capable of timing 1 hou

50
Q

ESR Reference Ranges

A

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
Q

Technical/Mechanical Factors
(Sources of Error) affecting an ESR

Tube position

A

u Tilt (3 degrees = 30% error)
u Vibration = Increases ESR
u Length and diameter of tube

52
Q

Technical/Mechanical Factors
(Sources of Error) affecting an ESR

Temp

A
  • Increased temperature = Increased ESR
  • Decreased temperature = Decreased ESR
53
Q

Technical/Mechanical Factors
(Sources of Error) affecting an ESR

Time

A

: >4 hours @ RT or >24 hours @ 4 degrees =
Decreases the ESR

54
Q

Technical/Mechanical Factors
(Sources of Error) affecting an ESR

3 others

A
  • Anticoagulant used - over-coagulation (too much
    anticoagulant)
  • Proper mixing of specimen
  • Presence of anemia – may be seen as
    pathological or physiological
55
Q

Quality Control of ESR

A
  • 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
Q

Label tubes for ESR

A
  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
Q

Reports

A

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
Q

Recap qustions to think

A
  • Define ESR
  • Define Zeta potential
  • Methodology
  • Sources of error
59
Q

Review from LO 1

A
  • 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
Q

ESR Review

A
  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
Q

Reporting
CSF
Colour
and
Clarity:
Review

A

Color:
* Colourless
* Yellow
* Pink
* Red
* Brown

Clarity:
* Clear
* Slightly Cloudy
* Cloudy

62
Q

Hemoglobin . . . characterics

A
  • Is a protein
  • Contains iron
  • Transports oxygen
  • Comprises ~ 35% of the cell volume
  • Is what makes blood red
63
Q

The function of Hemoglobin is to . . 2

A
  • Transport oxygen from the lungs to the body
  • Maintain body pH (pH inversely proportional to CO2)
64
Q

heme essential component

A

Ferrous (+2)
“Us” - carries oxygen

65
Q

storage iron heme

A

Ferritin (+3)

Ferric state = “Icky” – can not carry oxygen

66
Q

HEME portion of Hemoglobin 2 prts

A

*- One Atom of Ferrous Iron
*- One Mole of Oxygen

67
Q

Globin portion of Hemoglobin

A
  • Globin - has 4 amino acid chains
68
Q
  • 3 Different types of Hemoglobin present in a normal individual:
A

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
Q

Hemoglobin (HGB) evolution controlled by 2

A
  • Evolves: controlled by genetics and gestational age
70
Q

time and amounts of Embryonic hemoglobin → fetal hemoglobin → adult hemoglobin

A
  • 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
Q

3 steps in Measuring Hemoglobin

A

Release the hemoglobin from the RBC

Measure the hemolysate using a spectrophotometer

Hemoglobin is a STABLE colored product

72
Q

Principle of Spectophotometry

100% transmitted light =
0 =

A

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
Q

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

A

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
Q

Sodium Lauryl Sulfate Method

what is SLS
max absorbance of SLS
does it fallow beer law
2 advantages of SLS

A
  • 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
Q

4 prts of a Hemoglobin Curve

units
follows what law

samples at what absorbance

what are we reading off the graph?

A

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
Q

sample type for hemoglobin

A
  • Whole Blood
    • EDTA
    • Heparinized capillary
77
Q

hemoglobin Sources of Interference

A
  • 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
Q

Reference Ranges hemoglobin

A

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
Q

MICROHEMATOCRIT PRINCIPLE

hemocrit
defined as

A
  • 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
Q

microhematocrit The Method Equipment and Procedure

A

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
Q

Manual Hematocrit
Microhematocrit method calculation

A

RBC volume in mm / =20.0 mm/
Total volume in mm 59.0 mm

0.33898 L/ 1L = 0.339 L/L

82
Q

Technical Sources of Error for Manual Method hematocrit

A

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
Q

Physiological Sources of Error microhematocrit

A
  • 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
Q

Controlling Sources of Error microhematocrit

A
  • 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
Q

hematocrit Reference Ranges

Remember – this is
a test that NOW
has three decimals
when reporting

A

-Males 0.400 - 0.540 L/

-Females 0.330 - 0.480 L/L

-Newborns 0.400 - 0.620 L/L

86
Q

Clinical Significance
Increased in certain disease states:

A
  • Polycythemia
  • Dehydration - burn victims, severe diarrhea, vomiting
87
Q

Clinical Significance
decreased in certain disease states:

A
  • Anemias
  • Leukemias
  • Bleeding disorders
88
Q

clinical significant Hemorrhage - no change

A

Hemorrhage - no change for up
to 24 hours, then plasma volume
begins to increase to replace fluid
loss and dilution of blood occurs

89
Q

Specimen type microhematocrit

2 options

A
  • Good specimen/collection:
  • No hemolysis
  • No tissue fluid
  • EDTA
  • Heparin
  • Capillary (plain or heparin)
90
Q

Why EDTA in Hematology?

A
  • 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
Q

Heparin in Hematology?

A
  • 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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