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Fundamentals of MLS > Blood Smears > Flashcards

Blood Smears Flashcards

1
Q

What tubes should blood for smears be collected in?

A

Ethylenediaminetetraacetic acid (EDTA) lavender top preserves morphology best.

Note: Various tubes are used to collect samples for laboratory testing. The purple top tube which we use routinely for CBC testing contains EDTA which binds calcium and prevents clotting so individual cells can be accurately counted and evaluated

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2
Q

Why is it important to prepare and evaluate peripheral blood smears?

A
  1. Clinical significance: Provides valuable health information which can lead to proper diagnosis and treatment of many diseases.
  2. Components of the PBS evaluation
    a) White blood cell (WBC) estimates
    b) Platelet estimates
    c) Relative proportion of leukocyte types
    d) Cell morphologic structure of all 3 cell lines
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3
Q

How long after taking the blood should the blood smear be made and why?

A
  1. Time from draw to slide making within 4 hrs within 4 hours to minimize distortion due to EDTA.
  2. After 5 hrs. artifactual cell changes such as crenated RBCs, spherocytes, necrobiotic leukocytes, and vacuolated neutrophils. Vacuolation of monocytes occurs almost immediately with EDTA.
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4
Q

Why are tubes are used to collect samples for laboratory testing of blood smears?

A

Advantages are multiple slides can be made. Slides do not have to be made immediately.
Generally prevents platelets from clumping on slide

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5
Q

Do some people think capillary puncture should be the source of blood for blood smears?

A

Some purists believe anti-coagulant free blood such as from a fingerpoke still best choice as there is no anticoagulant effect on the morphology.

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6
Q

When is it recommended to use a Sodium Citrate tube for blood smears?

A

Occasionally a patient’s blood will contain antibodies which aggregate platelets in EDTA and interfere with platelet counts and blood smear evaluation. In these cases a blue top Sodium Citrate tube is drawn and used for such purposes.

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7
Q

When are heel and finger pokes used as sample locations for blood smears?

A

Heel pokes are commonly used to obtain blood from babies as obtaining venous blood can be difficult. Finger pokes can be used in adults in cases where obtaining venous is not possible.

Both heel pokes and finger pokes have their limitations.

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8
Q

What causes platelet satellitism and aggregation (clumping)? How can this be prevented?

A
  1. Both of these are caused by antibodies in the patient’s blood reacting in an EDTA collected sample which is not suitable for accurate counts or PBS evaluation.
  2. This interference can be removed by collecting patient’s blood in a Na Citrate tube and performing counts and PBS evaluation on the Na Citrate sample.
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9
Q

If using sodium citrate for CBC or peripheral blood smear tests how do the results need to be modified? Why?

A

Na Citrate tubes contain 0.5 ml. of anticoagulant with 4.5 ml. of blood when properly filled. When performing counts or estimates the dilutional effect must be taken into account and corrected. CBC results and Peripheral Blood Smear (PBS) **WBC and Platelet estimates must be multiplied by 1.1 before being reported.

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10
Q

What are heel and finger puncture limitations on blood smears?

A

Smears made directly from drops of blood may have platelet clumping if good flow is not achieved.
Only a few smears can be made.
Larger amounts can be collected with EDTA micro-collection tubes from the same site.

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11
Q

What is the most common technique for peripheral blood smear preparation?

A

Wedge blood smear technique.

Note: Attention to technique and detail is crucial to prepare a properly made PBS. A properly made PBS is absolutely essential to the proper reporting and interpretation of cell counts and morphologies.

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12
Q

What are the steps for the peripheral blood smear wedge technique?

A

STEPS

  1. Need two slides – one for the smear and one as a slider
  2. Apply one small drop on the slide close to frosted edge
  3. With the slider slide at a 30-45 degree angle back up the slider slide into the blood drop
  4. Pause to let the drop disperse along the slider (1 second)
  5. Push in one smooth motion across the slide
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13
Q

What are characteristics of a proper wedge blood smear?

A
  1. Size: the film is 2/3 to 3/4 the length of the slide
  2. Width: little narrower than slide (lateral edges are visible) finger shaped, slightly rounded at the feather edge, not bullet shaped
  3. Smooth distribution of blood ( no ridges, streaks, holes)
  4. Proper thickness (not too thick or thin) with suitable width to count and assess 100 WBCs, and cell morphology of all 3 cell lines
  5. Should use entire drop of blood.
  6. Smooth feathered edge, no jagged tails with rainbow appearance at feather edge when held up to the light
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14
Q

What are advantages and disadvantages of the automated technique for creation of blood smears?

A

Automated technique
Advantage: Can be connected to a CBC analyzer and Laboratory Information System (LIS) which allows PBS to be made automatically according to criteria.
Disadvantages: Cannot manually control the smear making process.

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15
Q

What do the procedures of automated blood smears taken into account?

A

Procedure: is dependent on the hematocrit reading, blood drop size, angle and speed is adjusted, then spreader slide is cleaned.

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16
Q

What methods are used for dying of blood smears?

A

Methods for drying may include small fan

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17
Q

What artifacts can occur if the blood smear is not dried properly?

A
  1. Drying of smears should be as quickly as possible

2. Artifacts: Slow drying can cause RBC’s to be crenated (shrunken, no central pallor, spiny projections).

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18
Q

Besides slow drying what are other reasons to cause crenated blood cells?

A

Slow drying, high humidity, blood >4 hrs. old, alkaline pH from glass slide, excess EDTA from underfilled tubes.

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19
Q

What are the three main steps to the blood smear staining process?

A

1st step: Apply Stain dissolved in a diluent such as methanol
2nd step: Apply Buffer of a specific pH 6.4 to 6.8
3rd step: Apply Rinse such as water or commercial rinse

Note: All PBS stains either use this 3 step method or a derivative of this 3 step method.
MEMORIZE THIS.

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20
Q

What are the components in a Romanowsky Stain?

A 
Romanowsky Stains (Polychrome stain)
Dye components are:
1.) Methylene blue (basic dye)
2.) Thiazines which are azure dyes- Azure A, B, C, and thionin are oxidative products of methylene blue
3.) Eosin (acid dye)
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21
Q

What are some commonly Romanowsky stains?

A

Commonly used Romanowsky stains include Wright’s, Wright/Giemsa, Leishman, May-Grunwald, Jenner’s (named after their developers)

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22
Q

What is a Romanowsky Stain?

A

The Romanowsky Stain is a polychrome stain (more than one stain) which contains a combination of methylene blue or derivatives of methylene blue and eosin. These stains which contain different colors and charges will stain cellular components different colors with great detail allowing for accurate identification and differentiation.

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23
Q

What is the purpose of the methanol in the Romanowsky stain?

A

Methanol -

  1. Acts as the solvent/diluent (Wrights’s stain which is a powder and needs to be diluted to a liquid)
  2. Fixes the slides the cells to the slide. Blood film would wash off if methanol was not used to fix the slide.
  3. Also methanol chemically prepares the blood for staining.
24
Q

What occurs in the buffer step? What pH range should the buffer be and why?

A
  1. Actual dissociation of the staining components and binding to the cellular structures takes place when the buffer is added.
  2. Buffer pH should be 6.4 to 6.8. as staining reactions are pH dependent. An acid or alkaline pH outside of these ranges dramatically affects staining quality.
25
Q

What is the purpose of the rinse solution?

A

The Rinse solution is simply to wash off the stain/buffer mixture after which drying occurs and the stained blood smear can be examined microscopically.

26
Q

What does methylene blue stain, i.e. name the cellular components? Is it basic or acidic?

A

Methylene blue is basic and stains acidic (basophilic) cellular components such as RNA and DNA nucleoproteins a blue color

27
Q

What cellular components does eosin stain and what colour? Is it acidic or basic?

A

Eosin is acidic and stains basic (eosinophilic) components such as hemoglobin and eosinophilic granules various combination of red and orange colors

28
Q

Why do neutrophils pick up staining characteristics of both Methylene blue and Eosin?

A

Neutrophils are so named as their granules have a neutral pH and pick up staining characteristic from both stains. This reaction is describes as neutrophilic.

29
Q

What does mean to have an azure-eosin complex and what is the result? What components does this affect?

A
  1. Methylene blue and eosin combine to form an azure-eosin complex inside the cell.
  2. Result: Stain some cellular components a purple color.
  3. Chromatin, some neutrophil granules, azurophilic granules, and platelets stain in this manner. This reaction is described as azurophilic.
30
Q

What are the advantages and disadvantages of the horizontal rack method of manual staining of smears?

A

Manual staining of smears
Procedure: horizontal rack method
Advantages: Timing can be increased for samples such as increased WBC count and bone marrow samples
Disadvantages: must be present, risk of spilling and longer time to complete

31
Q

What are the advantages and disadvantages of the ‘Quick stain’ method of manual staining of smears?

A

Manual staining of smears
Procedure: Quick stains
Advantages: fast and easy
Disadvantages: Quality is a concern. May have to adjust staining and buffering times

32
Q

What is another method to stain smears using separate jars?

A

Another method of staining is to put stains, buffer and rinse in separate coplin jars. Various types of Quick Stains use this method.

33
Q

What are the advantages and disadvantages of Hematek and CBC analyzer stainers used for automatic staining?

A

Automated staining of smears
Procedure: Hematek and CBC analyzer stainers
Advantages: High volume, walk away, good quality staining
Disadvantages: Cannot add stat smears to some batch stainers.

34
Q

What are the causes for blood smears that are too thick?

A

1) Too large drop of blood.
2) Spreading blood too quickly.
3) Spreader slide angle is too high.

35
Q

What problems are causes when the blood smear is too thick?

A

Red cells tend to form more rouleaux in thick smears.

WBC’s tend to stain darker in thick smears making identification difficult.

36
Q

What are the causes for blood smears that are too thin?

A

A) Too small a drop of blood
B) Spreading blood too slowly
C) Spreader angle too low

37
Q

What are four technical errors causing streaks in tail area of smear?

A

A.) Large numbers of leukocytes (but can’t be changed).
B.) Delay in spreading
C.) Rough or dirty spreader slide
D.) Using only part of drop of blood.

Low power inspection is important to detect smear and staining errors.

38
Q

What are causes for incorrect distribution of leukocytes?

A
  1. Result if entire drop of blood is not used
  2. Blood touching edge of glass slide cause larger cell (ie neuts, monos) to concentrate at edges.
  3. Smears made very slowly –> deposits larger cells at end and edges of smear.
  4. Very thin smears have increased number of lymphocytes in counting area.
  5. Ragged tailed ends of smears have higher number of larger cells in tails.
39
Q

What problems do thick smears cause in the analysis?

A

Very thick smears:

  1. Make identification of leukocytes and evaluation of RBC’s difficult because crowded so close together.
  2. Will not stain properly.
40
Q

What problems do too thin smears have for analysis?

A

Very thin smears may have broken cells and cell may be distorted in size, shape and staining.

41
Q

What type of blood films provide optimal cell morphology?

A

Fingerstick blood films provide optimal cell morphology.

42
Q

What are the steps of with Romanowsky stain?

A

STEP ONE:
Cells must be fixed to the slide with methanol
STEP TWO:
Addition of buffer solution changes pH, ionizes the reactants causing stain to dissociate.
STEP THREE:
A rinse solution (usually distilled H2O with proper pH or commercial soln.) is added to remove stain and stain residue.

43
Q

Will some staining occur during the first stage of fixation when Romanowsky (Wright’s) stain is used?

A

Minimal staining of elements may occur during the first stage of fixation-staining when the Romanowsky (Wright’s) stain is added to the slide but this step is mostly meant to fix the blood smear and saturate the blood smear with stain.

44
Q

What is the theory behind why the Romanowsky stain works when the buffer is added?

A

Addition of buffer solution changes pH, ionizes the reactants causing stain to dissociate. The acid dye eosin which is anionic or negatively charged is released. The basic dye ions (Methylene Blue, Azures A, B, C and thionin which are positively charged are also released to stain their oppositely charge cellular components
This is when most of the staining occurs

45
Q

What problems can result from inadequate fixation?

A

Inadequate fixation may result in: blood washing off slide, indistinct nuclear detail, poor staining of granules, especially basophils.

46
Q

What problem does water contamination of the methanol cause?

A

Water contamination of methanol may cause drying artifact.

47
Q

How long should the fixation period be?

A

Fixation period should be minimum of 1 minute.

48
Q

Why should tap water not be used as a substitute for buffer?

A

Tap water should NOT be used as substitute for buffer because: pH is usually wrong, frequently too alkaline or too acidic and may contain chlorine

49
Q

How long should the blood smear be in the buffer?

A

Staining time with buffer should be at least 2 times the fixation time.

50
Q

What can cause the smear to be too red (7 reasons)?

A
  1. Too acid a buffer or stain soln (pH below 6.4)
  2. Excess buffer for stain solution.
  3. Insufficient staining time
  4. Excessive washing
  5. Very thin smear
  6. Contaminants (ie chlorine) in wash H20.
  7. Exposure of buffer or stain solution to acid fumes.
    Old stain-Methanol oxidized to fumaric acid.
51
Q

What can cause too blue of a stain?

A
  1. Too alkaline a buffer or stain soln pH >6.8.
  2. Too little buffer for stain soln.
  3. Excessive staining time.
  4. Inadequate washing.
  5. Short drying time
  6. Wash H20 too alkaline
  7. Smears too thick.
  8. Old smear (dried plasma blue background)
  9. Protein abnormality (ie multiple myeloma)
  10. Heparin blood sample. Buffered Na Citrate
  11. Very high WBC count- Low Hematocrit
52
Q

What will the macroscopic colour look of a well-stained blood smear?

A

Macroscopic color: normal pink to purple

53
Q

How will the macroscopic colour look if the specimen is too old?

A

Color if specimen is old: too blue as fresh blood acts as buffer.

54
Q

How will the macroscopic colour look when there is increased proteins present?

A

Color when increased proteins present: bluer stained even if fresh sample as proteins stain blue.

55
Q

What are the microscopic colours of RBCs, WBC nuclei, Neutrophil cytoplasm, Eosinophil granules of a well-stained blood smear?

A

Microscopic color
RBCs: orange to salmon pink
WBC nuclei: purple to blue
Neutrophil cytoplasm: pink, violet, purple
Eosinophil granules: coarse, bright orange, refractile

56
Q

How are details represented on a well-stained blood smear?

A

Good detail of elements such as chromatin, nucleoli, granules, RBC inclusions.

57
Q

What is examined on a peripheral blood smear to ensure it is good?

A

Peripheral smear examination:

  1. Procedures performed
  2. Macroscopic examination:
    a) bluer than normal indicate proteins,
    b) grainy appearance cold agglutinins,
    c) holes may indicate high lipid.
    d) May require trouble shooting for automated CBC results.
  3. Markedly increased WBC and platelet counts may show blue specks at feather edge of smear.

Fundamentals of MLS (18 decks)

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