Retic analysis and Validation Flashcards

Question

Validation of Results:

Answer 1

Rule of 3 – Normal HCT should be 3X of the Hgb +/- 3 -Failure to meet this may indicate sample integrity issues or abnormal RBCs Linearity Limits – Results proportional to the calculated concentration/activity of the analyte -Samples above linear range – Sample must be diluted and results need to be multiplied by dilution factor Analyzer flags – Due to analyzer, specimen or patient abnormalities. Results must be investigated before release Nonsense Results – Results are so out of range it’s seen as incompatible with life (sometimes due to interfering substances) MCV > 130, MCH/MCHC greatly increased

Answer 2

Which parameters are directly measured? WBC Total Count and DIFF, RBC, PLT, Hgb PLT volume/ MCV on new analyzers Which parameters are calculated? MCV on Michener AcT5diff AL analyzers HCT L/L=[RBC(1012/L)x MCV (10-15L)] MCH (pg)= [Hb (g/L)/RBC (x1012/L] MCHC (g/L)= [Hb (g/L)/Hct L/L] RDW-CV: derived from RBC histogram WBC Absolute counts: derived from DIFF & Total WBC counts

Answer 3

How the analyzer counts cells ) Cells enter a conductive diluent 2) The counting chamber has a current applied between the external and internal electrode 3) When cell passes through aperture, this disturbs flow between internal and external electrode – Creates measurable voltage pulse 4) Oscilloscope displays pulses generated by the cells interrupting the current Number of Pulses is PROPORTIONAL to number of cells counted Size of Pulses is PROPORTIONAL to size of the cell RDW – Histogram showing size distribution of cells counted

Answer 4

Absorbance AcT5 - Tungsten halogen lamp New Technology- laser optical scatter AcT5- Forward scatter (FS): 0°to detect cell volume or size Side scatter (SS): 90° to detect internal complexity (granules, vacuoles etc.) Cytochemistry Diff Fix contains Chlorazole black Stains granules of Neutrophils, Lymphocytes, Monocytes & Eosinophils Volume Focused flow impedance- measures volume of cell (change in resistance as cell passes through flow cell aperture) Dual focused flow - where cells line up Has a 5 part differential Cytochemical staining Differential Lysis Technologies of Absorbance Technologies of Cytochemistry Coulter Impedance Principle

Answer 5

Based on Cell Size (Volume) and Complexity (Absorbance) Lymphocytes Small volume, low complexity/absorbance Neutrophils Medium volume, moderate complexity Monocytes Large volume, low complexity Eosinophils Medium volume, high complexity X-axis= Absorbance Y-axis= Volume

Answer 6

Electronic Impedance – Counting WBCs, RBCs and PLTs Cyanmethemoglobin Method– Hemoglobin Determination AcV Technology – WBC Diff Determination

Answer 7

counting and differentiating blood cells -composed of saline -stabilizes cell membranes for counting and sizing

Answer 8

Lyses erythrocytes. Fixes leukocytes. Differentially stains granules of monocytes, neutrophils, and eosinophils

Answer 9

Lyses red blood cells for the leukocyte count. Used to differentiate basophils.

Answer 10

Lyses red blood cells. Used to determine hemoglobin concentration.

Answer 11

Enzymatic solution with proteolytic action. Used as a rinsing agent.

Answer 12

A dilution is made, and a certain amount is removed and sent for dilution in RBC/PLT bath Remaining dilu’n is used for Hemoglobin Determination RBCs are diluted again and lysed with HB Lyse reagent Hbg released and is converted to cyanmethemoglobin and measured at 540 nm What could go wrong? -Lipemia or High WBC Count – Both increase turbidity which affects spectrophotometric reading of Hgb

Answer 13

Amount removed from First dilution/Hgb bath is then: Further diluted using Diluent reagent RBCs and PLTs are counted and discriminated by electrical impedance (Coulter principle) RBC count = All blips between 30-300 fL PLT Count = All blips up to 18 fL

Answer 14

Develop the RBC histogram, which is needed to obtain the HCT, MCV, and RDW results MCH calculated from RBC count and HGB value MCHC calculated from HGB value and HCT Develop the PLT histogram, which is needed to obtain MPV results

Answer 15

Total WBC count is performed -lyses RBCs and specifically differentiates between basophils and other leukocytes by volume Next basophils are counted Resistance of the basophils to the acidic lytic reagent is the basis for the differentiation of the basophils from the other leukocytes False cell counts? -nRBCs – resistant to lyse and can be counted as WBCs 100-400 fL – WBCs 300-400 fL – Likely basophils RBCs were lysed, PLTs are too small and won’t register on histogram (>20 fL)

Answer 16

-Very high WBC – They’re included in counts from 100-300 fL -Giant PLTs – Counted as over 30 fL

Answer 17

Very high WBC – may affect HCT and RBC – interfering substances -Very small RBCs (ex. Fragments >20 fL) – They aren’t counted as RBCs. So this can FALSELY INCREASE PLT counts -RBC agglutination – Increases RBC size so that it’s excluded from RBC count Falsely decreased MCV? -Giant PLTs – counted as small RBCs

Answer 18

Tight RDW, most cells are a uniform size. Decreased anisocytosis = 11.4% Large RDW indicates a large difference between smallest and largest cells therefore increased anisocytosis = 27

Answer 19

MCV RI = 80-100 fL Right Shift – Expect macrocytes Left shift – Expect microcytes Two Distinct Peaks – 2 populations of cells

Answer 20

Whole blood aliquot is mixed in DIFF bath with AcT5diff Fix reagent and Diluent -cells are lysed -Differentially stains Lymphocytes, Monocytes, Neutrophils, Eosinophils -Primary focused flow for impedance (volume) Second focused flow for optical detection (absorbance + cytochem)

Answer 21

Combined results obtained from WBC/BASO bath + DIFF bath

Answer 22

Turbidity affects spectrophotometric reading - Falsely Increased – Hgb, MCH -the instrument will show abnormal histrogram -Corrective Action – Plasma replacement

Answer 23

Increased turbidity affects spectrophotometric reading -Falsely Increased – Hgb, RBC count, -Incorrect HCT -Rule of 3 will not match -Corrective Action – Manual HCT, Correct RBC count, Recalculate RBC indices, Dilute the sample

Answer 24

clumps exclude it from being counted as RBC Falsely Decreases –RBCs, MCV Falsely increases – MCHC -has a grainy appearance -instrument can show right shift on RBC histrogram Corrective Action – Warm specimen to 37C + rerun

Answer 25

Falsely Decrease –PLT count Falsely Increases – WBC count -large clumps are counted as platelets Corrective Action IF PATHOLOGICAL - Redraw specimen in sodium citrate and multiply result by 1.1 Corrective Action IF COLLECTION ISSUE – Redraw sample in EDTA, the and do 8-10 inversions

Answer 26

Lyse resistant so it gets counted with WBCs Falsely Increase – WBC counts because nucleated RBCs are large so they are counted at WBC Corrective Action – Make PBF and count number of NRBCs per WBCs to correct WBC count

Answer 27

These are counted with PLTs bc so small Falsely Decreased – RBC count Falsely Increased – PLT count May see LEFT shift in RBC histogram Corrective Action – Look at PBF and confirm PLT estimate, then possible a manual PLT count

Answer 28

- Hct should be 3x the value of the Hgb (± 3) -3 x Hgb/1000 = Hct ± 0.03 L/L -for Normocytic/Normochromic erythrocytes -indicate abnormal RBCs or may be 1st indicator of error (sample integrity)

Answer 29

used more in clinical -compares current with most recent -values can stay the same unless there was major intervention -if the delta check fails this can indicate analytical error or mislabeled sample -must investigate , sample held until cause is found

Answer 30

Reticulocyte count-Increased polychromasia Iron studies - hypo/micro Sickle screen -Sickle cells Hbel - N/N anemia with targets, folded cells crystals +/- sickles Osmotic fragility -Spherocytes DAT-Spherocytes & polychromasia G6PD Assay-Bite/Blister cells