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Hematology > Weeks 5/6 > Flashcards

Weeks 5/6 Flashcards

Investigating Anemia, Lab Evaluation

1
Q

What should be done if the Rule of 3 is OK?

A

Examine other indices.

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2
Q

What is the next step if MCV is decreased?

A

Consider microcytic anemias – check MCH & MCHC.

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3
Q

What should be considered when MCV is increased?

A

Consider macrocytic anemias or increased polychromasia (↑ Poly).

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4
Q

What should you check if there are abnormal findings in MCV or other indices?

A

Check the RBC histogram for dual populations, shifts to the left or right, or micro RBCs.

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5
Q

What conditions could you consider if RBC count is normal?

A

Chronic and/or compensated condition.

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6
Q

What should be considered if RBC count is decreased?

A

Consider blood loss, acute hemolysis, iron deficiency anemia (IDA), megaloblastic anemia, etc.

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7
Q

What should you consider if RBC count is increased?

A

Consider thalassemia minor.

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8
Q

What should be considered if platelet (PLT) count is decreased?

A

Examine the sample, consider microangiopathic hemolytic anemia (MAHA), megaloblastic anemia, immune thrombocytopenic purpura (ITP), etc.

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9
Q

What conditions are associated with increased polychromasia in a hypochromic microcytic picture?

A

Treated iron deficiency anemia (IDA), Thalassemia, Hemoglobin E.

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10
Q

What conditions are associated with target cells in a hypochromic microcytic picture?

A

Thalassemia, Hemoglobin E.

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11
Q

What conditions are associated with inclusions or nucleated red blood cells (NRBCs) in a hypochromic microcytic picture?

A

Thalassemia, Hemoglobin E.

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12
Q

What condition is associated with the presence of sickle cells in a hypochromic microcytic picture?

A

Sickle-Thalassemia.

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13
Q

What tests would you perform to investigate a hypochromic microcytic picture?

A

Iron studies, Hemoglobin electrophoresis, and supravital staining.

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14
Q

What is the RDW, Serum Iron, TIBC, and Serum Ferritin in iron deficiency anemia?

A

RDW: Increased
Serum Iron: Decreased
TIBC: Increased
Serum Ferritin: Decreased

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15
Q

What are the typical lab values (RDW, Serum Iron, TIBC, Serum Ferritin) in α Thalassemia?

A

RDW: Normal
Serum Iron: Normal
TIBC: Normal
Serum Ferritin: Normal

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16
Q

What are the typical lab values in β Thalassemia?

A

RDW: Normal
Serum Iron: Normal
TIBC: Normal
Serum Ferritin: Normal
A₂ Level: Increased

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17
Q

What are the lab values for Hemoglobin E disease?

A

RDW: Normal
Serum Iron: Normal
TIBC: Normal
Serum Ferritin: Normal

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18
Q

What are the typical lab values in anemia of chronic disease?

A

RDW: Normal
Serum Iron: Decreased
TIBC: Decreased
Serum Ferritin: Increased

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19
Q

What are the typical lab values in sideroblastic anemia?

A

RDW: Increased
Serum Iron: Increased
TIBC: Decreased
Serum Ferritin: Increased

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20
Q

What are the typical lab values in lead poisoning?

A

RDW: Normal
Serum Iron: Normal
TIBC: Normal
Serum Ferritin: Normal
FEP: Increased

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21
Q

What is indicated by a normocytic/normochromic picture with increased polychromasia and many target cells, inclusions, and NRBCs?

A

Consider hemoglobinopathy (e.g., sickle cell disorders).

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22
Q

What tests would you perform to investigate a suspected hemoglobinopathy?

A

Sickle solubility test
Hemoglobin electrophoresis

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23
Q

What is indicated by a normocytic/normochromic picture with increased polychromasia, many spherocytes, and NRBCs?

A

Consider autoimmune hemolytic anemia (AIHA) or hereditary spherocytosis (HS).

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24
Q

What test would you perform to confirm the presence of spherocytes?

A

Osmotic fragility test.

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25
Q

What test would you perform to differentiate between AIHA and HS?

A

Direct Antiglobulin Test (DAT).

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26
Q

What specific features would you look for in a hemoglobinopathy?

A

Look for SC or C crystals or sickle cells.

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27
Q

What is the morphology and MCV in G6PD deficiency?

A

Normal MCV with blister/bite cells and normal or delayed increased polychromasia.

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28
Q

What is the morphology and MCV in Pyruvate Kinase (PK) deficiency or Paroxysmal Nocturnal Hemoglobinuria (PNH)?

A

Normal MCV with non-specific findings and normal or delayed increased polychromasia.

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29
Q

What is the morphology and MCV in Microangiopathic Hemolytic Anemia (MAHA)?

A

Normal MCV with fragments and normal or delayed increased polychromasia.

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30
Q

What tests should be performed when morphology shows bite cells, fragments, or non-specific findings?

A

Tests for intravascular hemolysis
Coagulation tests
Specific enzyme tests

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31
Q

What are key morphological features seen in G6PD deficiency?

A

Blister cells and bite cells.

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32
Q

What is the MCV and morphology in conditions such as burns or hemolysis?

A

MCV is normal, and morphology shows microspherocytes, RBC fragmentation, and budding.

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33
Q

What should you suspect if MCV is normal and microorganisms are seen on the smear?

A

Consider infections like malaria or other parasitic infections.

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34
Q

What are key tests to perform when malaria is suspected?

A

Speciation of the parasite
Determining the level of parasitemia
Rapid antigen test for malaria

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35
Q

What is a key consideration when interpreting abnormal RBC morphology in burns?

A

Look for patient history and perform tests for hemolysis.

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36
Q

What does normal or delayed increased polychromasia with microspherocytes and RBC fragmentation indicate?

A

Possible hemolysis or damage due to burns.

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37
Q

What is the first step when MCV is greater than 100 (macrocytic anemia)?

A

Check the smear, RDW, and RBC histogram.

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38
Q

What should you look for in the smear if you see oval macrocytes and hypersegmented neutrophils?

A

Consider Vitamin B12 deficiency, red cell folate deficiency, pancytopenia, and order tests such as Anti-IF antibodies, Schilling test, and bone marrow biopsy.

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39
Q

What should be tested if the smear shows round macrocytes and target cells?

A

Check liver enzymes for possible liver disease.

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40
Q

What does increased polychromasia on the smear indicate?

A

Increased polychromasia may indicate hemolytic anemia or a high reticulocyte count.

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41
Q

Why should you check the reticulocyte count in macrocytic anemia?

A

The high MCV could be due to reticulocytes, so checking the smear for other cell populations or signs of hemolytic anemia is necessary.

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42
Q

What type of test is the Sickle Solubility Screening Test for Hemoglobin S?

A

A qualitative screening test for Hemoglobin S.

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43
Q

What are the components of the reagent used in the Sickle Solubility Screening Test?

A

A high phosphate buffer
A hemolyzing agent (saponin)
A reducing agent (sodium dithionite)

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44
Q

What happens to Hemoglobin S during the Sickle Solubility Screening Test?

A

Hemoglobin S forms tactoids (crystals) in a reduced state.

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45
Q

What is the purpose of lysing RBCs in the Tube Solubility Screening Test for Hemoglobin S?

A

To free Hemoglobin S (Hb S) for testing.

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46
Q

What happens to Hemoglobin S when it is reduced in the Tube Solubility Screening Test?

A

It becomes insoluble, resulting in a turbid solution.

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47
Q

How is a positive result interpreted in the Tube Solubility Screening Test for Hemoglobin S?

A

Any presence of Hemoglobin S causes a positive result, indicated by turbidity in the solution.

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48
Q

What does a turbid solution indicate in the Tube Solubility Screening Test?

A

The presence of insoluble Hemoglobin S, which gives a positive result.

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49
Q

What is a common cause of a false negative result in the Sickle Solubility Test?

A

A low amount of Hemoglobin S (Hb S).

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50
Q

Why might newborns give a false negative result in the Sickle Solubility Test?

A

Newborns have a high amount of Hemoglobin F (Hb F), which can interfere with the detection of Hb S.

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51
Q

How can severe anemia affect the Sickle Solubility Test?

A

Patients with hemoglobin (Hb) levels less than 70 g/L may give a false negative result.

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52
Q

How do heterozygotes with Hb S less than 20% affect the Sickle Solubility Test?

A

They may result in a false negative due to insufficient Hb S concentration.

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53
Q

Why might recently transfused patients give a false negative result in the Sickle Solubility Test?

A

The transfusion dilutes the amount of Hb S in the bloodstream, potentially leading to a false negative.

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54
Q

What should be done if a low amount of Hb S is suspected in a sample?

A

Double the quantity of the sample to increase the likelihood of showing turbidity.

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55
Q

Why is the temperature of the reagent important in the Sickle Solubility Test?

A

The sickling process is temperature-sensitive, and a reagent that is too cold can cause a false negative.

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56
Q

What is the correct temperature for the reagent in the Sickle Solubility Test?

A

The reagent must be brought to room temperature (RT) before testing.

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57
Q

What are some common causes of false positives in the Sickle Solubility Test?

A

Hyperlipidemia/Lipemia
Extreme leukocytosis
Excess gamma globulins
Adding too much sample

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58
Q

Why might hyperlipidemia cause a false positive in the Sickle Solubility Test?

A

The increased lipids in the blood can interfere with the clarity of the solution, mimicking a positive result.

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59
Q

How can extreme leukocytosis affect the Sickle Solubility Test?

A

Excess white blood cells can cause turbidity in the solution, leading to a false positive.

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60
Q

What effect does the presence of excess gamma globulins have on the Sickle Solubility Test?

A

Excess gamma globulins can cause the solution to appear cloudy, resulting in a false positive.

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61
Q

How can adding too much sample affect the Sickle Solubility Test?

A

Adding too much sample can increase the likelihood of turbidity, leading to a false positive result.

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62
Q

Which non-Hb S hemoglobin variant can also give a false positive in the Sickle Solubility Test?

A

Hemoglobin C-Harlem, among other variants, can cause a false positive result.

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63
Q

Why must Hb S be confirmed after a positive Sickle Solubility Test result?

A

To ensure that the positive result is due to Hb S and not another hemoglobin variant or interfering factor.

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64
Q

What is the purpose of hemoglobin electrophoresis?

A

To separate proteins (hemoglobins) according to their charge.

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65
Q

How do normal hemoglobins behave in hemoglobin electrophoresis?

A

They show different mobility patterns in an electric field at a fixed pH.

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66
Q

How do variant hemoglobins differ in hemoglobin electrophoresis?

A

Amino acid substitutions in variant hemoglobins change the charge, affecting their mobility.

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67
Q

Why do some hemoglobins have the same mobility in hemoglobin electrophoresis?

A

Certain hemoglobins have the same mobility at specific pH levels due to their similar charge characteristics.

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68
Q

What is the pH used for routine screening in Alkaline Hemoglobin Electrophoresis?

A

Alkaline pH 8.4.

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69
Q

What buffer and gel are used in Alkaline Hemoglobin Electrophoresis?

A

Tris-EDTA-borate buffer and cellulose acetate gel.

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70
Q

What is the net charge of hemoglobin molecules at an alkaline pH in electrophoresis?

A

Hemoglobin molecules have a net negative charge.

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71
Q

Toward which electrode do hemoglobin molecules migrate in Alkaline Hemoglobin Electrophoresis?

A

Hemoglobin molecules migrate toward the anode (+).

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72
Q

Which hemoglobin type migrates the fastest in normal hemoglobin migration?

A

Hemoglobin A (Hb A) migrates the fastest, and nothing runs with Hb A.

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73
Q

How does Hemoglobin F (Hb F) migrate compared to Hemoglobin A (Hb A)?

A

Hemoglobin F (Hb F) migrates slightly behind Hemoglobin A.

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74
Q

Which hemoglobin type migrates the slowest in normal hemoglobin migration?

A

Hemoglobin A2 (Hb A2) migrates the slowest.

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75
Q

Which hemoglobins do not have any variants that run with them in alkaline electrophoresis?

A

Hemoglobins A and F.

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76
Q

Which hemoglobin variants run between Hemoglobin A and Hemoglobin A2 in alkaline electrophoresis?

A

Hemoglobins S, D, and G.

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77
Q

Which hemoglobin variants run with Hemoglobin A2 in alkaline electrophoresis?

A

Hemoglobins C, E, and O.

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78
Q

What should you assume if there is a heavy band at the A2 position in alkaline electrophoresis?

A

Assume it is one of the variants like Hemoglobin CC, SC, or EE, because A2 is never increased by more than approximately 10%.

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79
Q

What combination of normal and abnormal marker hemoglobins are used as purchased/commercial controls in Hemoglobin Alkaline Electrophoresis?

A

Hemoglobins A, F, S, and C.

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80
Q

Which hemoglobins are used as controls for a normal adult in Hemoglobin Alkaline Electrophoresis?

A

Hemoglobins A and A2.

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81
Q

Which hemoglobins are used as controls for a normal baby in Hemoglobin Alkaline Electrophoresis?

A

Hemoglobins F and A.

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82
Q

Which hemoglobins are faster than Hemoglobin A in electrophoresis?

A

Hemoglobin H and Hemoglobin Bart’s are the only hemoglobins faster than Hemoglobin A.

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83
Q

Are Hemoglobin H and Hemoglobin Bart’s typically detected on Hemoglobin Electrophoresis?

A

No, Hemoglobin H and Hemoglobin Bart’s are usually not detected on Hemoglobin Electrophoresis.

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84
Q

What is often required for abnormal hemoglobins found on cellulose acetate in alkaline hemoglobin electrophoresis?

A

Confirmation by other methods is usually required for abnormal hemoglobins.

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85
Q

Which hemoglobins run together with Hemoglobin S on cellulose acetate in alkaline electrophoresis?

A

Hemoglobin S runs with Hemoglobins D and G.

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86
Q

Which hemoglobins run together with Hemoglobin A2 on cellulose acetate in alkaline electrophoresis?

A

Hemoglobins C, E, and O run with Hemoglobin A2.

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87
Q

What hemoglobin separation technique is performed using acid agar?

A

Citrate (Acid) agar electrophoresis.

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88
Q

What advanced technique is commonly used for hemoglobin separation?

A

High Performance Liquid Chromatography (HPLC).

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89
Q

Which test is used to quantify Hemoglobin A2?

A

Hemoglobin A2 is quantified using ion exchange chromatography.

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90
Q

What staining technique is used to detect Hemoglobin H bodies?

A

Supravital staining.

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91
Q

What pH is used in Citrate Agar Hemoglobin Electrophoresis?

A

Citrate buffer at pH 6.0 (acidic pH).

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92
Q

What is the purpose of Citrate Agar Electrophoresis?

A

It is used to further fractionate hemoglobins that run together at alkaline pH.

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93
Q

Why is Citrate Agar Electrophoresis considered a confirmation test?

A

Too many abnormal hemoglobins run with Hemoglobin A, so it is not used as a primary test.

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94
Q

What is the recommended procedure when using electrophoresis to separate hemoglobins?

A

Always run Alkaline Electrophoresis first, then acid (Citrate Agar) electrophoresis if needed for confirmation.

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95
Q

What hemoglobins run together at alkaline pH in electrophoresis?

A

Hemoglobins S, D, G, and Hemoglobins C, E, O, A2.

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96
Q

What hemoglobins are separated by Citrate Agar Acid Electrophoresis?

A

Hemoglobin C is separated from Hemoglobins E and O.
Hemoglobin S is separated from Hemoglobins D and G.

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97
Q

What can cause poor separation, artifacts, or smearing of hemoglobin bands in electrophoresis?

A

Improper loading of the sample, such as trapped air or peeling.

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98
Q

How can improper blotting affect hemoglobin electrophoresis results?

A

If the blotting is too wet or too dry, it can lead to poor separation or smearing of hemoglobin bands.

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99
Q

What effect does excessive heat during electrophoresis have on the results?

A

It can cause smearing or artifacts in the hemoglobin bands.

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100
Q

Why should the application points not be placed too close to the anode in hemoglobin electrophoresis?

A

Application points that are too close to the anode can lead to poor separation of hemoglobin bands.

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101
Q

What issues can arise from delays in applying current, application of the sample, or staining after electrophoresis?

A

Delays can result in artifacts, poor separation, or smearing of hemoglobin bands.

102
Q

What can cause problems with hemoglobin electrophoresis aside from loading issues?

A

Problems with reagents or equipment.

103
Q

How can sample quality affect hemoglobin electrophoresis results?

A

Poor samples can lead to inaccurate results.

104
Q

What can happen if the wrong pH or ionic strength is used in hemoglobin electrophoresis?

A

It can result in poor separation or incorrect band formation.

105
Q

How do contaminated or cloudy buffers affect hemoglobin electrophoresis?

A

They can cause artifacts and distort the results.

106
Q

Why is it important to ensure that wells, applicator tips, and cellulose acetate plates are clean?

A

Contamination with dirt, blood, or other proteins can interfere with the results.

107
Q

How does High Performance Liquid Chromatography (HPLC) compare to traditional methods in hemoglobin analysis?

A

HPLC is much more automated.

108
Q

Which hemoglobins can be detected using HPLC?

A

Hemoglobins S, F, A, C, D, and E can be detected and measured accurately based on quantitative thresholds.

109
Q

What is a limitation of HPLC in distinguishing certain hemoglobins?

A

HPLC cannot distinguish Hemoglobin A2 from Hemoglobin E well.

110
Q

For which hemoglobins is HPLC commonly used for quantitation?

A

HPLC is commonly used for quantitating Hemoglobins F and A2.

111
Q

What type of column is utilized in High Performance Liquid Chromatography (HPLC) for hemoglobin analysis?

A

A weak cation exchange column.

112
Q

How are hemoglobins retained in the HPLC column?

A

Hemoglobins are retained based on their charge when injected into a buffered resin column.

113
Q

What happens to hemoglobin variants during HPLC?

A

Hemoglobin variants are eluted (come off the column) at a particular retention time.

114
Q

How are different hemoglobins identified in HPLC?

A

Different peaks are identified in defined windows, and their retention time, relative percentage, and area are printed out.

115
Q

How do amino acid substitutions in hemoglobin variants affect HPLC results?

A

They alter the retention time relative to Hemoglobin A.

116
Q

What advantage does automated HPLC provide compared to manual methods?

A

It offers a more efficient and accurate assessment of Hemoglobin A2 and Hemoglobin F.

117
Q

What is the retention time for Hemoglobin F in HPLC?

A

1.15 minutes.

118
Q

What is the retention time for Hemoglobin A in HPLC?

A

2.60 minutes.

119
Q

What is the retention time for Hemoglobin A2 in HPLC?

A

3.63 minutes.

120
Q

What is the retention time for Hemoglobin E in HPLC?

A

3.69 minutes.

121
Q

What is the retention time for Hemoglobin D in HPLC?

A

4.10 minutes.

122
Q

What is the retention time for Hemoglobin S in HPLC?

A

4.50 minutes.

123
Q

What is the retention time for Hemoglobin C in HPLC?

A

5.20 minutes.

124
Q

What are the retention times for Hemoglobin Bart’s (γ₄) and Hemoglobin H (β₄) in HPLC?

A

0.2 minutes for both.

125
Q

What is typically observed in Hemoglobin A2 levels in cases of β-thalassemia minor using HPLC?

A

Increased Hemoglobin A2 levels.

126
Q

Why is HPLC preferred over hemoglobin electrophoresis for quantifying Hemoglobin A2?

A

HPLC is more accurate at quantifying Hemoglobin A2 than hemoglobin electrophoresis.

127
Q

What advantage does HPLC have in separating hemoglobins compared to hemoglobin electrophoresis?

A

HPLC can separate Hemoglobin A2 from certain hemoglobins, which is not possible using hemoglobin electrophoresis alone.

128
Q

What pH range is used in the gel support for Isoelectric Focusing?

A

pH range of 3-10.

129
Q

What happens to hemoglobins during Isoelectric Focusing?

A

Hemoglobins find their isoelectric point, which is the point of no net negative charge.

130
Q

What is a key feature of the bands observed in Isoelectric Focusing?

A

Very distinct bands with all hemoglobins separated.

131
Q

Why is Isoelectric Focusing more expensive compared to other methods?

A

Due to the cost of materials and the technology required, it is a more expensive technique.

132
Q

In what type of screening is Isoelectric Focusing commonly used?

A

Newborn screening.

133
Q

What is a unique advantage of Isoelectric Focusing in detecting hemoglobin abnormalities?

A

It can pick up small amounts of abnormal hemoglobin.

134
Q

What are small protein carrier ampholytes used for in Isoelectric Focusing?

A

They are incorporated in the support medium, such as agarose, to help establish the pH gradient.

135
Q

What happens when current is applied during Isoelectric Focusing?

A

The ampholytes migrate and establish a desired pH gradient throughout the gel.

136
Q

What happens to proteins or hemoglobins in the gel during Isoelectric Focusing?

A

They migrate to their isoelectric points, which is the pH at which their net charge is zero.

137
Q

Why do hemoglobin variants separate on the gel during Isoelectric Focusing?

A

Due to differences in their isoelectric points.

138
Q

What does the position of Hemoglobin A, F, and S indicate in a normal vs. sickle cell trait vs. sickle cell anemia pattern?

A

Normal: Hb A is dominant.
Sickle Cell Trait: Both Hb A and Hb S are present.
Sickle Cell Anemia: Hb S is dominant, with no Hb A present.

139
Q

What is Capillary Electrophoresis?

A

A technique that separates sample components based on the difference in charge-to-size ratio through an electric field.

140
Q

How is the capillary prepared for electrophoresis?

A

It is filled with a conductive fluid at a certain pH, and voltage is applied.

141
Q

What happens to sample components in Capillary Electrophoresis?

A

They migrate through the capillary at different speeds based on their charge-to-size ratio.

142
Q

What role does the Electro-Osmotic Flow (EOF) play in Capillary Electrophoresis?

A

EOF is a force stronger than the electrical field, causing all proteins to be carried towards the cathodic end of the capillary.

143
Q

What voltage is typically applied in Capillary Electrophoresis?

A

A high voltage, up to more than 300 V/cm, is applied to create the electric field for separation.

144
Q

How are hemoglobin fractions detected in Capillary Electrophoresis?

A

Hemoglobin fractions are displayed as peaks, and the system automatically marks the zones where the variants belong.

145
Q

What is an advantage of Capillary Electrophoresis over other techniques?

A

It can separate and quantify Hb A2 in the presence of Hb E.

146
Q

What is similar between Capillary Electrophoresis and HPLC?

A

Both techniques display hemoglobin fractions as peaks for detection.

147
Q

What technique is used to separate Hemoglobin A₂ in column chromatography?

A

Anion-exchange chromatography (resin).

148
Q

What is the role of the resin in Hemoglobin A₂ column chromatography?

A

The resin is a preparation of cellulose covalently coupled to small positively charged molecules.

149
Q

How does the positively charged cellulose in the resin function in the separation process?

A

It attracts and binds negatively charged hemoglobin molecules.

150
Q

What factors are controlled to cause different hemoglobins to have different net charges in column chromatography?

A

The buffer and pH.

151
Q

How are hemoglobins selectively removed from the resin during column chromatography?

A

By altering the pH.

152
Q

Does Hemoglobin A₂ bind to cellulose in column chromatography?

A

No, Hb A₂ does not bind to the cellulose and is eluted as the reagent moves through the column. Hb A stays bound.

153
Q

What is measured before and after the elution process in Hemoglobin A₂ column chromatography?

A

The absorbance of the total hemoglobin fraction before the column, and then the A₂ fraction after elution.

154
Q

What is column chromatography used for in hemoglobin analysis?

A

It is used to separate Hb A₂ from HbC or HbE and to quantify Hb A₂.

155
Q

Which disorder is Hemoglobin A₂ quantification particularly important for?

A

β-thalassemia minor.

156
Q

What is the formula for calculating Hb A₂ percentage?

A

Hb A₂ % = (Abs of Hb A₂ Fraction / 5 x Abs of TF Solution) x 100

157
Q

What does the “Abs of Hb A₂ fraction” represent?

A

The absorbance of the solution (patient lysate) after it passes through the column (only the Hb A₂ fraction).

158
Q

What does the “Abs of TF solution” represent?

A

The absorbance of the solution (patient lysate) prior to elution through the column. This represents the total hemoglobin fraction.

159
Q

What is the significance of the number 5 in the Hb A₂ calculation formula?

A

The number 5 is the dilution factor of the specific column, where the total fraction tube is 15 mL, and the Hb A₂ fraction is in a 3 mL tube.

160
Q

What is the expected range for normal adult Hemoglobin A₂ (Hb A₂)?

A

Normal adult Hb A₂ is less than 3.5%.

161
Q

What does an Hb A₂ value between 3.5% and 8.0% indicate?

A

It is indicative of β-thalassemia trait.

162
Q

What does an Hb A₂ value greater than 8.0% indicate?

A

It indicates the presence of other abnormal hemoglobin variants.

163
Q

What is the most probable clinical condition associated with the presence of bite cells?

A

G6PD Deficiency

164
Q

What is the appearance of diffuse basophilia in supravital stain and its associated conditions?

A

Appearance: Dark blue granules and filaments in cytoplasm (seen in reticulocytes).
Associated conditions: Hemolytic anemia, after treatment for iron, vitamin B12, or folate deficiency.

165
Q

What is the composition of basophilic stippling and its associated conditions?

A

Composition: Precipitated RNA.
Associated conditions: Lead poisoning, thalassemias, hemoglobinopathies, megaloblastic anemia, myelodysplastic syndromes.

166
Q

What is the appearance of Howell-Jolly bodies in Wright stain and their composition?

A

Appearance: Dark blue-purple dense, round granule; usually one per cell, occasionally multiple.
Composition: DNA (nuclear fragment).

167
Q

What are Heinz bodies and in which conditions are they found?

A

Heinz bodies are round, dark blue-purple granules attached to the inner RBC membrane (seen in supravital stain).
Associated conditions: G6PD deficiency, unstable hemoglobins, oxidant drugs/chemicals.

168
Q

What are Pappenheimer bodies and their composition?

A

Appearance: Irregular clusters of small, light to dark blue granules, often near the periphery of the cell.
Composition: Iron.

169
Q

What is the appearance of Cabot rings in Wright stain and their composition?

A

Appearance: Blue rings or figure-eights.
Composition: Mitotic spindle remnant.

170
Q

What are Hb H inclusions, their appearance, and associated conditions?

A

Appearance: Fine, evenly dispersed dark blue granules giving a “golf ball” appearance to RBCs (seen in supravital stain).
Associated conditions: Hb H disease.

171
Q

What is Hereditary Persistence of Fetal Hemoglobin (HPFH)?

A

It is a rare disorder caused by a mutation in the β gene cluster that leads to slightly reduced β globin production and increased γ globin, resulting in elevated levels of fetal hemoglobin (Hb F).

172
Q

How much Hb F is typically seen in individuals with HPFH?

A

About 15% Hb F is often seen in individuals with HPFH.

173
Q

Is HPFH usually benign?

A

Yes, HPFH is usually benign unless it is inherited with a Hb S gene, in which case it can have clinical implications.

174
Q

What does the Betke-Kleihauer Acid Elution Test detect?

A

It detects Hemoglobin F or fetal RBCs in maternal circulation.

175
Q

What is the clinical application of the Betke-Kleihauer Test for Rh-negative mothers?

A

It is used to test Rh-negative mothers’ blood for the presence of Rh-positive fetal cells.

176
Q

How is the Betke-Kleihauer test related to Hereditary Persistence of Fetal Hemoglobin (HPFH)?

A

It is occasionally used for detecting HPFH.

177
Q

What happens to Hemoglobin A (Hb A) in the Betke-Kleihauer test?

A

Hb A dissolves out of solution.

178
Q

What happens to Hemoglobin F (Hb F) in the Betke-Kleihauer test?

A

Hb F is resistant to the acid medium and stains pink.

179
Q

What is the procedure for preparing the Betke-Kleihauer Acid Elution Test?

A

Make a peripheral blood smear (PBS) and incubate it in acid buffer.

180
Q

What counterstain is used in the Betke-Kleihauer test?

A

Eosin is used to counterstain the sample.

181
Q

How do fetal red blood cells appear in the Betke-Kleihauer Acid Elution Test?

A

Fetal red blood cells stay bright pink.

182
Q

How do maternal red blood cells appear in the Betke-Kleihauer Acid Elution Test?

A

Maternal red blood cells lose hemoglobin and appear as ghost cells.

183
Q

What is the cause of Paroxysmal Nocturnal Hemoglobinuria (PNH)?

A

PNH is caused by an acquired mutation of glycosylphosphatidylinositol (GPI)-anchored proteins, such as CD55 and CD59.

184
Q

What role does CD55 play in PNH?

A

CD55, also known as decay-accelerating factor, disrupts the formation of C3- and C5-convertase, protecting cells from complement-mediated lysis.

185
Q

What role does CD59 play in PNH?

A

CD59 is a membrane inhibitor of reactive lysis that prevents the formation of the membrane attack complex (MAC), thus protecting cells from lysis.

186
Q

Why are red blood cells in PNH susceptible to hemolysis?

A

Due to the lack of GPI-anchored proteins like CD55 and CD59, red blood cells in PNH are susceptible to complement activation and hemolysis.

187
Q

What is the Sucrose Hemolysis Test used for?

A

It is a screening test for suspected PNH patients and for any hemolytic anemia of unknown origin.

188
Q

What is added to red blood cells in the Sucrose Hemolysis Test?

A

A low-salt solution containing sucrose (sugar) is added to the red cells.

189
Q

What happens to complement in the Sucrose Hemolysis Test?

A

Complement is activated and binds to the red cells.

190
Q

Why are PNH red blood cells unusual in the Sucrose Hemolysis Test?

A

PNH red blood cells are unusually susceptible to complement, causing them to lyse.

191
Q

What is the result of the Sucrose Hemolysis Test in PNH patients?

A

PNH cells lyse, indicating increased fragility, whereas normal cells do not lyse.

192
Q

What is Ham’s Test or the Acidified Serum Test used for?

A

It is a screening test for Paroxysmal Nocturnal Hemoglobinuria (PNH).

193
Q

Why are RBCs of PNH patients sensitive in Ham’s Test?

A

RBCs of PNH patients are complement sensitive.

194
Q

What happens to RBCs at a slightly acidic pH in Ham’s Test?

A

Complement will affix to the RBCs, become activated by the alternate pathway, and result in the lysis of the RBCs.

195
Q

What is the purpose of heat-inactivated serum in Ham’s Test?

A

It serves as a control to show that no hemolysis occurs without active complement.

196
Q

What is the primary method used for confirming Paroxysmal Nocturnal Hemoglobinuria (PNH)?

A

Flow Cytometry to detect decreased or absent CD55 and/or CD59 markers.

197
Q

Which markers are analyzed in flow cytometry for PNH diagnosis?

A

CD55 and CD59.

198
Q

What technology is used to detect CD55 and CD59 in PNH confirmation?

A

Flow Cytometry with monoclonal antibodies bound to fluorescent dyes.

199
Q

What type of plot is produced in flow cytometry for PNH?

A

A scatterplot showing normal red cells and GPI-deficient red cells.

200
Q

What is the significance of GPI-deficient red cells in flow cytometry?

A

They indicate the absence or reduction of CD55/CD59, which is diagnostic for PNH.

201
Q

What does a scatterplot of fluorescence in flow cytometry indicate in PNH confirmation?

A

It indicates cell populations based on antigen detection.

202
Q

In flow cytometry for PNH confirmation, what does a high fluorescence level (populations circled in red on the right) signify?

A

It signifies the presence of a large amount of antigen, which is normal.

203
Q

What is the significance of populations that are high in fluorescence in flow cytometry for PNH?

A

These populations indicate that the cells have a normal level of antigen detection.

204
Q

In the context of PNH flow cytometry, what does a population with low or absent fluorescence indicate?

A

It may indicate a deficiency of GPI-anchored proteins, such as CD55 or CD59, which is characteristic of PNH.

205
Q

What does low scatter or fluorescence in flow cytometry indicate in PNH confirmation?

A

It indicates decreased antigens on the red blood cells.

206
Q

In PNH confirmation by flow cytometry, what do populations on the left side of the scatterplot represent?

A

These populations represent cells with decreased antigen levels, which is characteristic of GPI-deficient red cells in PNH.

207
Q

What can flow cytometry detect in granulocytes of PNH patients?

A

Flow cytometry can detect the absence of GPI-linked proteins (like CD55 and CD59) and FLAER expression in granulocytes.

208
Q

Why are WBCs preferred over RBCs for flow cytometry in PNH confirmation?

A

WBCs are more reliable because they are not affected by recent transfusions, making them easier to use for flow cytometry.

209
Q

Which white blood cells are used to detect PNH through flow cytometry?

A

Neutrophils (Neuts) and Monocytes (Monos) can be used to detect the same defect in flow cytometry.

210
Q

What is the purpose of the Gel Agglutination Assay for PNH?

A

It detects CD 55 (DAF) and CD 59 (MIRL) antigens using specific monoclonal antibodies.

211
Q

What does a positive reaction in the Gel Agglutination Assay indicate?

A

A positive reaction confirms the presence of MIRL or DAF, denoting that the patient does not have PNH.

212
Q

What does a negative result or double population in the Gel Agglutination Assay indicate?

A

Negative results or double population confirm the presence of PNH due to a lack of antigens (Ag).

213
Q

What process is followed in the Gel Agglutination Assay to confirm PNH?

A

After centrifugation, cells carrying antibodies for MIRL or DAF will show a positive reaction if present, indicating no PNH. If absent, it indicates PNH.

214
Q

What enzyme catalyzes the reaction in the Pyruvate Kinase Activity Assay?

A

Pyruvate kinase.

215
Q

In the second step of the Pyruvate Kinase Activity Assay, what happens to the pyruvic acid formed in the first step?

A

Pyruvic acid reacts with NADH (high fluorescence) to form lactic acid and NAD (no fluorescence) through lactic dehydrogenase.

216
Q

What is the significance of fluorescence in the Pyruvate Kinase Activity Assay?

A

High fluorescence is seen with NADH, and no fluorescence indicates the conversion to NAD.

217
Q

What is incubated with the patient’s red blood cells during the Pyruvate Kinase Activity Assay?

A

The red blood cells are incubated with reagents containing ADP, phosphoenolpyruvic acid, and NADH.

218
Q

What happens to fluorescence in a normal Pyruvate Kinase (PK) activity assay?

A

Fluorescence disappears within 15 minutes.

219
Q

What is the significance of persistent fluorescence in a PK activity assay?

A

It indicates Pyruvate Kinase (PK) deficiency, where pyruvic acid is not produced, and fluorescence persists for 45 to 60 minutes.

220
Q

What happens in Pyruvate Kinase (PK) deficiency during the PK activity assay?

A

Pyruvic acid is not produced, and the fluorescence persists for a longer duration (45 to 60 minutes).

221
Q

In the PK activity assay, which enzyme catalyzes the conversion of pyruvic acid and NADH into lactic acid and NAD?

A

Lactic dehydrogenase.

222
Q

What is the expected result for a normal G6PD activity assay?

A

Fluorescence is observed.

223
Q

What does the absence of fluorescence in the G6PD activity assay indicate?

A

G6PD deficiency.

224
Q

Why should the G6PD test be done after a hemolytic episode has resolved?

A

Reticulocytes (retics) have higher levels of G6PD, which can give a false normal result during a hemolytic episode.

225
Q

What reaction is catalyzed by the G6PD enzyme in the activity assay?

A

Glucose-6-phosphate + NADP → 6-Phosphogluconate + NADPH (with fluorescence).

226
Q

During a hemolytic crisis, what staining technique is used to detect Heinz bodies in G6PD deficiency?

A

Supravital stain is used to stain the patient’s blood, and a smear is made to detect Heinz bodies.

227
Q

What do Heinz bodies indicate when observed in a blood smear during G6PD deficiency?

A

Heinz bodies represent denatured hemoglobin and are associated with oxidative damage to red blood cells during a hemolytic crisis in G6PD deficiency.

228
Q

What is the characteristic appearance of Heinz bodies in a G6PD deficiency blood smear?

A

Heinz bodies appear as small, round inclusions within red blood cells when stained with a supravital stain.

229
Q

What does the osmotic fragility test measure in red blood cells?

A

It measures the surface area to volume ratio of red blood cells (RBCs).

230
Q

What happens when whole blood is added to a series of graded hypotonic salt solutions in the osmotic fragility test?

A

Water enters the red blood cells to maintain osmotic equilibrium, causing the cells to swell and become spherical.

231
Q

How do RBCs behave in a hypotonic solution during the osmotic fragility test?

A

RBCs swell as water enters the cells, and in extreme hypotonic solutions, all cells may lyse (burst).

232
Q

What is the appearance of RBCs in an isotonic solution during the osmotic fragility test?

A

RBCs maintain a normal biconcave shape and remain intact.

233
Q

What is the expected result of RBCs in a hypertonic solution in the osmotic fragility test?

A

RBCs shrivel and become crenated due to water leaving the cells.

234
Q

What happens when a critical volume is reached in red blood cells during the osmotic fragility test?

A

The cell contents leak out, causing the cell to burst, and hemoglobin is released.

235
Q

How is hemoglobin release detected in the osmotic fragility test?

A

The released hemoglobin can be measured with a spectrophotometer.

236
Q

How do red blood cells of patients with hereditary spherocytosis (HS) behave in the osmotic fragility test compared to normal cells?

A

HS patients have less tolerance for swelling, and their red blood cells lyse at higher salt concentrations, closer to isotonic solutions.

237
Q

At what saline concentration do spherocytes start to lyse in the osmotic fragility test?

A

Spherocytes start to lyse as soon as the saline concentration drops below isotonic levels.

238
Q

At what concentration of NaCl does initial hemolysis for normal erythrocytes begin in the osmotic fragility test?

A

Initial hemolysis for normal erythrocytes begins at 0.45 - 0.55% NaCl.

239
Q

At what concentration of NaCl is complete hemolysis observed in normal erythrocytes in the osmotic fragility test?

A

Complete hemolysis occurs at 0.38% NaCl.

240
Q

What does increased fragility indicate in the osmotic fragility test?

A

Increased fragility indicates hemolysis starting at 0.85% or 0.65% NaCl, which is lower than the normal range.

241
Q

What is reported in the osmotic fragility test besides the concentration of NaCl at which hemolysis starts?

A

The percentage of complete hemolysis is also reported, indicated by a pink supernatant.

242
Q

What does normal fragility indicate in the osmotic fragility test?

A

Normal fragility indicates hemolysis starting around 0.55% NaCl.

243
Q

What does an increase in spherocytes indicate in the osmotic fragility test?

A

An increase in spherocytes indicates an increase in osmotic fragility, as seen in conditions like Hereditary Spherocytosis (HS) and Autoimmune Hemolytic Anemia (AIHA).

244
Q

What condition is associated with a decrease in osmotic fragility due to an increase in target cells?

A

Thalassemias are associated with decreased osmotic fragility due to an increase in target cells.

245
Q

What chemistry tests are used to differentiate between intravascular and extravascular hemolysis?

A

Haptoglobin, Hemoglobinuria, and Bilirubin.

246
Q

Which laboratory test differentiates between Iron Deficiency Anemia (IDA) and Thalassemia?

A

Iron studies.

247
Q

What microbiology test is used to differentiate between Microangiopathic Hemolytic Anemias (MAHAs) like Hemolytic Uremic Syndrome (HUS)?

A

Bacterial identification.

248
Q

What is the purpose of Perl’s Prussian Blue stain in histotechnology?

A

It is used to stain iron in urine sediment and is positive in intravascular hemolysis but not in extravascular hemolysis.

249
Q

Which transfusion test differentiates between Hereditary Spherocytosis (HS) and Autoimmune Hemolytic Anemia (AIHA)?

A

Direct Antiglobulin Test (DAT).

250
Q

How do you confirm Hemolytic Disease of the Fetus and Newborn (HDFN) for ABO or Rh incompatibility?

A

By using the DAT, mother’s antibody identification, and baby’s DAT & eluate.

Hematology (6 decks)

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