Hematology Lab Practical #1

Describe an adequately prepared peripheral blood smear.

The smear should:

Cover 2/3 of the slide

Feather shaped

Take the width of the slide w edges visible

How long can a blood smear be made from an EDTA tube at room temperature?

No more than 5 hours

What happens to blood in EDTA tubes if left at room temperature for more than 5 hours.

Red cells appear to be echinocytes

White cells can develop denatured or necrobiotic nuclei and vacuoles in the cytoplasm

Platelets can clump or agglutinate

How do you perform a WBC estimate?

Scan aprox 6-8 fields in good area of peripheral smear

Count WBCs and average based on #of fields.

At 10x multiply WBC seen by 200

If 40x multiply WBC seen by 2,000

Reported as WBC/uL

EX 10X: 34+31+33+37+32+35 = 202/6 = 33.67 X 200 = 6,733.33 WBC/uL

When performing a WBC estimate if examining the slide at 10x what do you multiply your avg # of fields by?

200x

When performing a WBC estimate if examining the slide at 40x what do you multiply your avg # of fields by?

2000x

How do you perform a Platelet Estimate?

Performed at 100X

Scan approx. 5-10 fields and count the number of Platelets

Multiple the average number of Platelets seen by 20,000

Reported as PLT/uL

Ex: Avg: 15+18+13+17+20= 83/5 = 17 x 20,000 = 340,000 platelets per uL

What power do you use for a PLT estimate? What do you multiply your count by?

100x

Multiply the average by 20,000

PLT/uL

What is the height and width of the hemacytometer?

3mm

What is the size of the large squares on the hemacytomer?

1mm

What is the depth of the hemacytometer?

0.1mm

What is the total volume of the hemacytometer?

9mm2 x 0.1mm = 0.9mm3

Where are the white cells counted on a hemacytometer?

What are the dimensions of this area?

The 4 white corners of the hemacytometer.

The dimensions are 1mm^2

Where are the platelets counted on a hemacytometer?

What are the dimensions of this area?

The center square (all 25 smaller squares)

1mm^2

Where are the red cells counted on a hemacytometer?

What are the dimensions of this area?

The center square (all 25 smaller squares)

1mm^2

What is the formula for a manual WBC cell count calculation?

#cells x dil factor x depth (10)

_________________

Areas counted

= WBC/uL

During a manual WBC cell count - how close should the counts be on each side of the chamber?

+/- 10%

If WBC counts are 14 or less does the +/- 10% rule apply?

No

Correction for presence of Nucleated RBC in WBC count formula

Corrected WBC = (uncorrected WBC x 100) / (# nRBC + 100)

When does a corrected WBC count need to be preformed?

When there are 5 or more NRBCs on the differential regardless of age

What is the normal amount of nucleated red blood cells in an adult? In a newborn?

Adult: 0

Newborn: <25 per 100

Normal WBC Count

3.6-10.6 x 10^3 / uL

Normal RBC count for adults male/femal

Male: 4.2-6 x 10^6 / uL

Female: 3.8-5.2 x 10^6 / uL

Normal Platelet Count

150,000-450,000 / uL

Normal HGB for adult male/female

Male: 13.5-18 g/dL

Female: 12-15 g/dL

Normal HCT for adult male/female

Male: 40-54%

Female: 35-49%

Normal MCV range

80-100 fL

Normal MCH

26-34 pg

Normal MCHC

32-38 %

Relative % of Neutrophil bands and segs in WBC differential

Bands 0-5%

Segs 50-70%

Relative % of Lymphocytes in WBC differential

18-42%

Relative % of Monocytes in WBC differential

2-11%

Relative % of Eosinophils in WBC differential

1-3%

Relative % of Basophils in WBC differential

0-2%

Calculation of absolute values

WBC x % cell line = cells/uL

Ex: WBC count of 2.0 x 10^3 / uL

80 neutrophils

absolute 2,000 x .80 = 1,600 neutrophils / uL

How is Neutrophenia / Neutrophilia or Lymphopenia / Lymphophilia determined?

Using the calculation of abosolute values.

In the previous example the PT has relative neutrophilia because of the relatively high percentage of neutrophils.

However, the absolute count is below reference range so the PT has absolute neutrophilia

Normal WBC Identification

A.Normal RBC

B.Segmented Neutrophil

C.Banded Neutrophil

D.Eosinophil

E.Basophil

F.Lymphocyte

G.Monocyte

H.Platelet

Ovalocyte + cause

altered membrane

Sickle Cell + cause

Abnormal globin chain structure caused by 1 single amino acid rearrangement

Acanthocyte + cause

Increased cholesterol

alphabetical less spikey

Echinocyte + cause

cation imbalance, change in toxicity

Schistocyte + cause

fragmented by fibrin

shistocyte! its the devil! fragmented by fibrin

Dacrocyte/teardrop cell + cause

Englarged spleen

dont cry about your enlarged spleen

Codocyte + cause

Liver disease

Howell-Jolly body + content + conditions

DNA

Seen post-splenectomy, megaloblastic anemias, hemolytic anemias

HOLLY JOLLY CHRISTMAS W/O UR ****IN SPLEEN

Reticulocyte

RNA (R for RNA)

associated w decreased RBC survival/hemorrage, erythroid hyperplastic marrow

Pappenheimer bodies

Iron

seen in sideroblastic anemia, thalassemia

prussian blue stain

Heinz Body + content + conditions

Precipitated hemoglobin (H for hemoglobin)

Seen in G6PD deficiency

Only seen w supravital state

Ketchup is wright = cant see them w wright stain need supravital stain

Hemoglobin C crystals

Crystalized Hgb C

Ringed Sideroblasts

Iron

Basophilic Stippling

RNA aggregates

Thalassemia and lead poisoning

Nucleated RBC

DNA

Calculation of MCV

MCV = Hct/RBC x 10

Calculation of MCH

HGB/RBC x 10

Calculation of MCHC

HGB/HCT x 100

HCT calculation fix

Hct = MCV x RBC

Anemia Classification MCV < 80 fL

Microcytic Anemia

Anemia Classification MCV > 100 fL

Macrocytic Anemia

Anemia Classification MCV 80-100 fL

Normocytic Anemia

GO OVER ANEMIA SLIDE MOAR

Osmotic Fragility Test normal values!

Normal Cells begin lysis at 0.45-0.55% NaCL

Osmotic Fragility

Has an increased osmotic fragility test

Lysis begins at >0.5% NaCL

Spherocytes have decreased surface to volume ratio. Cannot stand intake of excess water. Will lyse at higher concentrations of NaCL than normal RBC.

Target cells have increased surface area to volume ratio. Handles larger influx of water. Lysis occurs at lower NaCl concentration. Seen in hemoglobinopathies

Hemoglobin solubility test

Good screening tool

Deoxygenated Hgb S has decreased solubility when added to reducing agent

If Hgb solubility test is positive then?

Hemoglobin Electrophoresis

What is the screening tool in Hgb solubility test? What does it do?

Sodium dithionite - converts ferrous iron to ferric iron. Oxygen molecule becomes deoxygenated - polymerization

Causing turbidity

Hemoglobin electrophoresis

memorize order? know??? from video

Hemoglobin Electrophoresis Citrate agar

Separates C from E and S from DGE

Kleihaur-betke

Adult HGB elutes from RBC due to citrate acid buffer.

HGB F resists eluation and remains.

Kleihauer Betke Test

Calculation

% Hgb F = (total # of fetal cells/500) x 100

When are increased Hgb F values seen?

Hereditary persistence of hemoglobin F

Thalassemia major (40-90% Hgb F)

Thalassemia Minor (5-10% Hgb F)

Sickle Cell Anemia

Normal Hemoglobin F Levels

At birth

12 Weeks

4-5 Months

2y-adult

Birth: 60-90%

12 Wks: 7%

4-5M: 1.1-5.3%

2y-adult: 1-2%

Reticulocyte Count

# of retics counted x 100 = % reticulocytes

generally 10 fields which would be 100 RBCs per field

Normal Retic range

0.5-2.5%

Absolute retic count

Actual number of reticulocytes in 1 liter or 1 uL of blood

(% retics x rbc count)/100 retics/uL

Expected retic count value in each anemia

??

Retics

Low value before treatment

Higher value after treatment

Corrected retic counts

In specimens with a low Hct, the percentage of reticulocytes may be falsely elevated because the whole blood contains fewer red blood cells. A correction factor is used with the nromal Hct is considered 45%

Correctted retic count

% retic X (patient Hct / 45 = corrected retic count

Reference interval Retic

Patients w Hct of 35% should have elevated retic of 2-3%

Hct is >25% 3-5%

Corrected retic count is dependent on the degree of the anemia and determines how well the bone marrow is compensating for the anemia